Cytoskeleton A. Overview B. Experimental Methods C. Microtubules D. Microfilaments (Updated 4/9/08) A. Overview B. Experimental Methods C. Microtubules

CytoskeletonCytoskeletonA.A. OverviewOverview

B.B. Experimental MethodsExperimental Methods

C.C. MicrotubulesMicrotubules

D.D. MicrofilamentsMicrofilaments

(Updated 4/9/08)(Updated 4/9/08)

A. Overview

B. Experimental Methods

C. Microtubules

D. Microfilaments

A.A. OverviewOverview1.1. DefinitionDefinition

2.2. Types of Cytoskeleton FibersTypes of Cytoskeleton Fibers

3.3. Dynamic Dynamic Polymerization/DepolymerizationPolymerization/Depolymerization

4.4. Molecular MotorsMolecular MotorsAlberts:Alberts: Fig. 16 – 1, Panel 16 – 1, Panel 16 – 2, Fig. 16 –11, Fig 16 – 12, 16 – 8, 16 – 7, 16 – 10, 16 – 13, 16 – 14, 16 – 15, Fig. 16 – 1, Panel 16 – 1, Panel 16 – 2, Fig. 16 –11, Fig 16 – 12, 16 – 8, 16 – 7, 16 – 10, 16 – 13, 16 – 14, 16 – 15,

16 – 16, 16 – 17, 16 – 19, 16 – 56, Table 16 – 1 16 – 16, 16 – 17, 16 – 19, 16 – 56, Table 16 – 1

A. Overview


C. Microtubules

D. Microfilaments

B.B. Experimental methodsExperimental methods1.1. Visualization ApproachesVisualization Approaches

– Light MicroscopyLight Microscopy– Fluorescence MicroscopyFluorescence Microscopy

» http://http://www.itg.uiuc.edu/exhibits/gallery/fluorescencegallery.htmwww.itg.uiuc.edu/exhibits/gallery/fluorescencegallery.htm

– Digital/video MicroscopyDigital/video Microscopy– Electron MicroscopyElectron Microscopy

2.2. Genetic ApproachesGenetic Approaches

3.3. Biochemical ApproachesBiochemical Approaches

A. Overview


C. Microtubules

D. Microfilaments

C.C. MicrotubulesMicrotubules

1.1. StructureStructure

2.2. Microtubule-associated proteinsMicrotubule-associated proteins

3.3. FunctionsFunctions

4.4. Microtubule motorsMicrotubule motors

5.5. Microtubule organizing centersMicrotubule organizing centers

6.6. Dynamic properties of microtubulesDynamic properties of microtubules

7.7. Flagella and ciliaFlagella and cilia

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions

4. Microtubule Motors

5. MTOCs

6. Dynamic Properties

7. Flagella and Cilia

D. Microfilaments

C.1. Microtubules: StructureC.1. Microtubules: Structure

1.1. StructureStructure– Alberts: Fig 16 – 11 Alberts: Fig 16 – 11 – Structure and composition - hollow, tubular; found in Structure and composition - hollow, tubular; found in

most eukaryotic cells (cilia, spindle, flagella)most eukaryotic cells (cilia, spindle, flagella)» Outer diameter - 24 nmOuter diameter - 24 nm» Wall thickness - ~5 nmWall thickness - ~5 nm» May extend across cell length/breadthMay extend across cell length/breadth

– Wall composed of globular proteins arranged in Wall composed of globular proteins arranged in longitudinal rows (protofilaments)longitudinal rows (protofilaments)

– Protofilaments are aligned parallel to tubule long axis Protofilaments are aligned parallel to tubule long axis – In cross section, consist of 13 protofilaments arrayed In cross section, consist of 13 protofilaments arrayed

in circular pattern within wallin circular pattern within wall

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

– Each protofilament is assembled of dimeric building Each protofilament is assembled of dimeric building blocks (one blocks (one -tubulin & one -tubulin & one -tubulin; A heterodimer) -tubulin; A heterodimer) organized in linear array along length of protofilamentorganized in linear array along length of protofilament

– Two types of tubulin subunits have similar 3D structure & Two types of tubulin subunits have similar 3D structure & fit tightly togetherfit tightly together

– Protofilaments asymmetric (Protofilaments asymmetric (-tubulin at one end, -tubulin at one end, --tubulin at other); All in single MT have same polarity; tubulin at other); All in single MT have same polarity; Each assembly unit has 2 nonidentical components Each assembly unit has 2 nonidentical components (heterodimer)(heterodimer)

– All protofilaments of microtubule have same polarity; All protofilaments of microtubule have same polarity; Thus so does full tubule (plus- & minus-end)Thus so does full tubule (plus- & minus-end)

– Plus end - fast-growing (Plus end - fast-growing (-tubulins on tip); Minus end - -tubulins on tip); Minus end - slow-growing (slow-growing (-tubulins on tip)-tubulins on tip)

C.1. Microtubules: StructureC.1. Microtubules: Structure

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

2.2. Microtubule-associated proteinsMicrotubule-associated proteins– Alberts: Fig 16-40, 16-41Alberts: Fig 16-40, 16-41– MTs can assemble in vitro from purified tubulin, but MTs can assemble in vitro from purified tubulin, but

MAPs are found with MTs isolated from cells; most MAPs are found with MTs isolated from cells; most found only in brain tissue; MAP4 has wider found only in brain tissue; MAP4 has wider distribution distribution

– Have globular head domain that attaches to MT side Have globular head domain that attaches to MT side & filamentous tail protruding from MT surface& filamentous tail protruding from MT surface

– May interconnect MTs to help form bundles (cross-May interconnect MTs to help form bundles (cross-bridges), increase MT stability, alter MT rigidity, bridges), increase MT stability, alter MT rigidity, influence MT assembly rateinfluence MT assembly rate

C.2. Microtubules: MAPsC.2. Microtubules: MAPs

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 2. Microtubules: MAPsC. 2. Microtubules: MAPs– MAP activity controlled by addition & removal of MAP activity controlled by addition & removal of

phosphate groups from particular amino acid residues phosphate groups from particular amino acid residues by protein kinases & phosphatases, respectively; by protein kinases & phosphatases, respectively; example - Alzheimer’s disease (AD)example - Alzheimer’s disease (AD)

– Abnormally high MAP (tau) phosphorylation Abnormally high MAP (tau) phosphorylation implicated in fatal neurodegenerative diseases like implicated in fatal neurodegenerative diseases like AD; neurofibrillary tangles in brains made of AD; neurofibrillary tangles in brains made of hyperphosphorylated tau; may help kill nerve cellshyperphosphorylated tau; may help kill nerve cells

– Excessively phosphorylated tau molecules are unable Excessively phosphorylated tau molecules are unable to bind to MTs; people with one of these diseases, a to bind to MTs; people with one of these diseases, a type of dementia called FTDP-17, carry mutations in type of dementia called FTDP-17, carry mutations in tau gene, implicating it as causetau gene, implicating it as cause

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 3. Microtubules: FunctionsC. 3. Microtubules: Functions

3.3. FunctionsFunctions– Alberts: Table 16-2; Fig 16-23, 66Alberts: Table 16-2; Fig 16-23, 66

Internal skeleton (scaffold) providing structural Internal skeleton (scaffold) providing structural support & maintaining organelle positionsupport & maintaining organelle position

– Resist compression or bending forces on fiber; Resist compression or bending forces on fiber; provide mechanical support like girders in `tall provide mechanical support like girders in `tall building; prevent distortion of cell by cytoplasmic building; prevent distortion of cell by cytoplasmic contractionscontractions

– MT distribution conforms to & helps determine cell MT distribution conforms to & helps determine cell shape: flattened, round cells - radiate from nuclear shape: flattened, round cells - radiate from nuclear area; columnar epithelium - parallel to cell long axis; area; columnar epithelium - parallel to cell long axis; like aluminum rods support tentlike aluminum rods support tent

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 3.C. 3. Microtubules: FunctionsMicrotubules: Functions

– Elongated cell process (axon, axopods of heliozoan Elongated cell process (axon, axopods of heliozoan protists) - MTs oriented parallel to each other & axon protists) - MTs oriented parallel to each other & axon or axopod long axis; help move thingsor axopod long axis; help move things

– In developing embryo, extend growing central NS In developing embryo, extend growing central NS axons to peripheral NS; inhibit (colchicine [CO], axons to peripheral NS; inhibit (colchicine [CO], nocodazole [NO]) & outgrowth stops, regresses nocodazole [NO]) & outgrowth stops, regresses (collapses back to rounded cell body)(collapses back to rounded cell body)

– Found as core of axopodial processes of heliozoan Found as core of axopodial processes of heliozoan protozoa; many MTs arranged in spiral with protozoa; many MTs arranged in spiral with individual MTs traversing entire length of processindividual MTs traversing entire length of process

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 3. Microtubules: FunctionsC. 3. Microtubules: Functions Plants: play similar role in plants; affect shape Plants: play similar role in plants; affect shape

indirectly by influencing cell wall formation; indirectly by influencing cell wall formation; found in cortex just below membrane during found in cortex just below membrane during interphase forming a distinct cortical zoneinterphase forming a distinct cortical zone

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


Also have role in maintenance of cell internal Also have role in maintenance of cell internal organization (organelle placement) - disrupt MTs organization (organelle placement) - disrupt MTs (CO, NO) —> Golgi disperses to cell periphery; (CO, NO) —> Golgi disperses to cell periphery; goes back to cell center when inhibitors removedgoes back to cell center when inhibitors removed

Move macromolecules & organelles around cell Move macromolecules & organelles around cell in directed manner (intracellular motility) in directed manner (intracellular motility) – Halt vesicle transport between compartments if Halt vesicle transport between compartments if

disrupt MTs so transport dependent on themdisrupt MTs so transport dependent on them

– Proteins made in neuron cell body move down axon Proteins made in neuron cell body move down axon (neurotransmitters, etc.) in vesicles(neurotransmitters, etc.) in vesicles

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


– Different materials move at different rates; fastest rate is 5 Different materials move at different rates; fastest rate is 5 µm/sec (400 mm/day); vesicles seen attached to MTsµm/sec (400 mm/day); vesicles seen attached to MTs

– Structures & materials moving toward neuron terminals are Structures & materials moving toward neuron terminals are said to move anterograde said to move anterograde

– Other structures, like endocytic vesicles that are formed at Other structures, like endocytic vesicles that are formed at neuron terminals & carry regulatory factors from target cells, neuron terminals & carry regulatory factors from target cells, move from synapse to cell body in a retrograde directionmove from synapse to cell body in a retrograde direction

– Ex.: axons filled with MTs, MFs & IFs; evidence suggests Ex.: axons filled with MTs, MFs & IFs; evidence suggests that both anterograde & retrograde movement are mediated that both anterograde & retrograde movement are mediated mostly by MTs; video microscopy shows vesicles moving mostly by MTs; video microscopy shows vesicles moving along MTsalong MTs

– Confirmed by EM of axons; molecular motors move vesicles Confirmed by EM of axons; molecular motors move vesicles along the MTs that serve as tracksalong the MTs that serve as tracks

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


Motile elements of cilia & flagella (more Motile elements of cilia & flagella (more later)later)

Active components of mitotic/meiotic Active components of mitotic/meiotic machinery; move chromosomesmachinery; move chromosomes

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 4. Microtubules: MotorsC. 4. Microtubules: Motors

4.4. Microtubule motorsMicrotubule motors– Alberts: Fig 16-58, 59, 60, 62, 63, 64, 67Alberts: Fig 16-58, 59, 60, 62, 63, 64, 67

Motor proteins: convert chemical energy stored Motor proteins: convert chemical energy stored in ATP into mechanical energy that is used to in ATP into mechanical energy that is used to move cellular cargo attached to motormove cellular cargo attached to motor

– Types of cellular cargo transported by these Types of cellular cargo transported by these molecular motors include: vesicles, organelles molecular motors include: vesicles, organelles (mitochondria, lysosomes, chloroplasts), (mitochondria, lysosomes, chloroplasts), chromosomes, other cytoskeletal filamentschromosomes, other cytoskeletal filaments

– A single cell may contain dozens of different motor A single cell may contain dozens of different motor proteins, each specialized for moving a particular proteins, each specialized for moving a particular type of cargo in particular cell regiontype of cargo in particular cell region

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 4. Microtubules: MotorsC. 4. Microtubules: Motors– Collectively, motor proteins are grouped into 3 broad Collectively, motor proteins are grouped into 3 broad

families: myosins, kinesins, dyneinsfamilies: myosins, kinesins, dyneins» Kinesins & dyneins move along MTs; myosins move along Kinesins & dyneins move along MTs; myosins move along

MFs; None known for ifsMFs; None known for ifs» Motor proteins move unidirectionally along their Motor proteins move unidirectionally along their

cytoskeletal track in a stepwise manner from one binding cytoskeletal track in a stepwise manner from one binding site to the nextsite to the next

» As they move along, they undergo a series of As they move along, they undergo a series of conformational changes (a mechanical cycle)conformational changes (a mechanical cycle)

» Steps of mechanical cycle are coupled to chemical cycle, Steps of mechanical cycle are coupled to chemical cycle, which provides energy fueling movement which provides energy fueling movement

» Includes motor binding ATP, ATP hydrolysis, product Includes motor binding ATP, ATP hydrolysis, product (ADP & Pi) release & binding of new ATP(ADP & Pi) release & binding of new ATP

» Binding & hydrolysis of 1 ATP moves motor a few nm Binding & hydrolysis of 1 ATP moves motor a few nm along track; Cycles repeated many timesalong track; Cycles repeated many times

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


KinesinsKinesins– Kinesins move vesicles/organelles from cell body to Kinesins move vesicles/organelles from cell body to

synaptic knobs; isolated in 1985 from squid giant synaptic knobs; isolated in 1985 from squid giant axons; tetramer made of 2 identical heavy chains & 2 axons; tetramer made of 2 identical heavy chains & 2 identical light chains; smallest & best understoodidentical light chains; smallest & best understood

– Large protein - pair of globular heads generate force Large protein - pair of globular heads generate force by hydrolyzing ATP & bind MT; each head connected by hydrolyzing ATP & bind MT; each head connected to a neck, a rodlike stalk & fan-shaped tail that binds to a neck, a rodlike stalk & fan-shaped tail that binds cargo to be hauledcargo to be hauled

– Diverse superfamily of kinesins - heads similar since Diverse superfamily of kinesins - heads similar since roles similar; tails vary since they haul different roles similar; tails vary since they haul different cargoescargoes

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 4. Microtubules: MotorsC. 4. Microtubules: Motors– In vitro mobility assay - kinesin-coated beads move to In vitro mobility assay - kinesin-coated beads move to

MT "+" end (axon tip); it is a "+" end-directed MT MT "+" end (axon tip); it is a "+" end-directed MT motor, therefore, kinesin responsible for anterograde motor, therefore, kinesin responsible for anterograde movementmovement» All MTs of axon are oriented with"-" ends facing cell body & All MTs of axon are oriented with"-" ends facing cell body &

"+" ends facing synaptic knobs "+" ends facing synaptic knobs » Moves through ATP-dependent cross-bridge cycle along Moves through ATP-dependent cross-bridge cycle along

single MT protofilament (rate proportional to [ATP]; up to ~1 single MT protofilament (rate proportional to [ATP]; up to ~1 µm/sec); at low concentrations, move slowly & see µm/sec); at low concentrations, move slowly & see movement in distinct stepsmovement in distinct steps

» Each step is ~8 nm in length, the spacing between tubulin Each step is ~8 nm in length, the spacing between tubulin dimers along protofilament dimers along protofilament

» Appear to move 2 globular subunits (or 1 dimer at a time); Appear to move 2 globular subunits (or 1 dimer at a time); usually toward membrane & "+" endsusually toward membrane & "+" ends

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 4. Microtubules: MotorsC. 4. Microtubules: Motors– Kinesin possesses 2 motor domains that work by Kinesin possesses 2 motor domains that work by

“hand-over-hand” mechanism; one always firmly “hand-over-hand” mechanism; one always firmly attached to MTattached to MT» 2 heads of kinesin behave in coordinated manner, so that they 2 heads of kinesin behave in coordinated manner, so that they

are always present at different stages in their chemical & are always present at different stages in their chemical & mechanical cycles at a given timemechanical cycles at a given time

» When one head binds to MT, the interaction induces a When one head binds to MT, the interaction induces a conformational change in adjacent neck region of motor conformational change in adjacent neck region of motor protein; it swings the other head forward toward binding site protein; it swings the other head forward toward binding site on next dimeron next dimer

» Force generated by head catalytic activity leads to swinging Force generated by head catalytic activity leads to swinging movement of neckmovement of neck

» A kinesin molecule walks along a MT, hydrolyzing one ATP A kinesin molecule walks along a MT, hydrolyzing one ATP with each stepwith each step

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


Conventional kinesin (discovered in 1985) is only Conventional kinesin (discovered in 1985) is only one member of a superfamily of related kinesinsone member of a superfamily of related kinesins– Mammalian genome sequence analysis leads to Mammalian genome sequence analysis leads to

estimate that mammals make >50 different kinesinsestimate that mammals make >50 different kinesins

– Heads of kinesins have related amino acid sequences, Heads of kinesins have related amino acid sequences, reflecting common evolutionary ancestry & their reflecting common evolutionary ancestry & their similar role in moving along MTssimilar role in moving along MTs

– In contrast, kinesin tails have diverse sequences, In contrast, kinesin tails have diverse sequences, reflecting variety of cargo different proteins haulreflecting variety of cargo different proteins haul

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 4. Microtubules: MotorsC. 4. Microtubules: Motors– Most kinesins travel toward the "+" end; but one small Most kinesins travel toward the "+" end; but one small

subfamily of kinesins (including the Drosophila Ncd subfamily of kinesins (including the Drosophila Ncd protein) moves toward the MT "-" end protein) moves toward the MT "-" end » one would expect that the heads of "+"- & "-"-directed would one would expect that the heads of "+"- & "-"-directed would

have a different structure since the heads contain the catalytic have a different structure since the heads contain the catalytic core of the motor domain core of the motor domain

» But the heads are virtually indistinguishable; instead But the heads are virtually indistinguishable; instead differences in direction of movement are determined by differences in direction of movement are determined by differences in the adjacent neck regions of the two proteinsdifferences in the adjacent neck regions of the two proteins

» When the head of a "-" end-directed Ncd molecule is joined to When the head of a "-" end-directed Ncd molecule is joined to the neck-stalk portions of a kinesin molecule, the hybrid the neck-stalk portions of a kinesin molecule, the hybrid protein moves toward the "+" end of a MT trackprotein moves toward the "+" end of a MT track

» Even if the hybrid has a catalytic domain that would normally Even if the hybrid has a catalytic domain that would normally move toward the "-" end of a MT, as long as it is joined to the move toward the "-" end of a MT, as long as it is joined to the neck of a "+" end motor, it moves in the "+" directionneck of a "+" end motor, it moves in the "+" direction

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


A third subfamily of kinesinlike proteins is A third subfamily of kinesinlike proteins is incapable of movement: kinesins of this incapable of movement: kinesins of this group, like KXKCM1, are thought to group, like KXKCM1, are thought to destabilize MTs rather than acting as MT destabilize MTs rather than acting as MT motorsmotors

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


Cytoplasmic DyneinsCytoplasmic Dyneins– Dyneins - first MT-associated motor found (1963); Dyneins - first MT-associated motor found (1963);

responsible for moving cilia & flagellaresponsible for moving cilia & flagella– Thought to be ubiquitous eukaryotic motor protein; Thought to be ubiquitous eukaryotic motor protein;

related protein found in variety of nonneural cellsrelated protein found in variety of nonneural cells– Cilia/flagella form of protein was called axonemal Cilia/flagella form of protein was called axonemal

dynein; its new relatives were called cytoplasmic dyneindynein; its new relatives were called cytoplasmic dynein– Huge protein (~1.5 million daltons); 2 identical heavy Huge protein (~1.5 million daltons); 2 identical heavy

chains & variety of intermediate & light chainschains & variety of intermediate & light chains– Each dynein heavy chain forms large globular head Each dynein heavy chain forms large globular head

(~10X larger than a kinesin head) that generates force; (~10X larger than a kinesin head) that generates force; moves along MT toward "-" endmoves along MT toward "-" end

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 4. Microtubules: MotorsC. 4. Microtubules: Motors Suggested roles of cytoplasmic dyneinSuggested roles of cytoplasmic dynein

– Force generating agent for chromosome movement in Force generating agent for chromosome movement in mitosis mitosis

– "-"-directed MT motor for Golgi complex positioning "-"-directed MT motor for Golgi complex positioning & movement of vesicles/organelles through cytoplasm& movement of vesicles/organelles through cytoplasm

– In nerve cells, cytoplasmic dynein involved in axonal In nerve cells, cytoplasmic dynein involved in axonal retrograde organelle movement (toward cell body & retrograde organelle movement (toward cell body & cell center) & anterograde movement of MTscell center) & anterograde movement of MTs

– Fibroblasts & other nonneural cells: may move varied Fibroblasts & other nonneural cells: may move varied membranous organelles (endosomes, lysosomes, ER-membranous organelles (endosomes, lysosomes, ER-derived vesicles going toward Golgi) from periphery derived vesicles going toward Golgi) from periphery toward cell center toward cell center

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 4. Microtubules: MotorsC. 4. Microtubules: Motors– Cytoplasmic dynein does not interact directly with Cytoplasmic dynein does not interact directly with

membrane-bounded cargo, but requires intervening membrane-bounded cargo, but requires intervening multisubunit complex, dynactin that may regulate multisubunit complex, dynactin that may regulate dynein activity & help bind it to MTdynein activity & help bind it to MT

– Present model may be overly simplistic: kinesin & Present model may be overly simplistic: kinesin & cytoplasmic dynein move similar materials in cytoplasmic dynein move similar materials in opposite directions over the same railway networkopposite directions over the same railway network

– Individual organelles may bind kinesin & dynein Individual organelles may bind kinesin & dynein simultaneously although only one is active at given simultaneously although only one is active at given time; myosin may also be present on some of these time; myosin may also be present on some of these organellesorganelles

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 5. Microtubules: MTOCSC. 5. Microtubules: MTOCS

5.5. Microtubule-organizing centersMicrotubule-organizing centers– Alberts: Panel 16-1; Fig 16-29, 30, 31, 32, 33 Alberts: Panel 16-1; Fig 16-29, 30, 31, 32, 33

Function of MT in living cell depends on its Function of MT in living cell depends on its location & orientation, thus it is important to location & orientation, thus it is important to understand why a MT assembles in one place understand why a MT assembles in one place as opposed to anotheras opposed to another

– controlled by MT-organizing centers controlled by MT-organizing centers (MTOCs)(MTOCs)

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 5. Microtubules : MTOCSC. 5. Microtubules : MTOCS

Assembly of MTs from Assembly of MTs from -dimers occurs -dimers occurs in 2 distinct phasesin 2 distinct phases– Nucleation - slower; small portion of MT Nucleation - slower; small portion of MT

initially formed; occurs in association with initially formed; occurs in association with specialized structures in vivo called specialized structures in vivo called microtubule-organizing centers (MTOCs); microtubule-organizing centers (MTOCs); centrosome is examplecentrosome is example

– Elongation - more rapidElongation - more rapid

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


Centrosomes - complex structure with 2 barrel-Centrosomes - complex structure with 2 barrel-shaped centrioles surrounded by amorphous, shaped centrioles surrounded by amorphous, electron dense pericentriolar material (PCM)electron dense pericentriolar material (PCM)– In animal cells, cytoskeleton MTs typically form in In animal cells, cytoskeleton MTs typically form in

association with centrosomeassociation with centrosome

– Centrioles: cylindrical; ~0.2 nm dia & typically ~twice Centrioles: cylindrical; ~0.2 nm dia & typically ~twice as long; usually with 9 evenly spaced fibrilsas long; usually with 9 evenly spaced fibrils

– Each fibril seen in cross section to be composed of 3 Each fibril seen in cross section to be composed of 3 fused MTs (A, [the only complete one]; B & C), A is fused MTs (A, [the only complete one]; B & C), A is attached to centriole center by radial spokeattached to centriole center by radial spoke

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 5. Microtubules : MTOCSC. 5. Microtubules : MTOCS– 3 MTs of each triplet arranged in pattern that gives 3 MTs of each triplet arranged in pattern that gives

centriole a characteristic pinwheel appearancecentriole a characteristic pinwheel appearance

– Centrioles usually in pairs at right angles to each other Centrioles usually in pairs at right angles to each other near cell center just outside nucleusnear cell center just outside nucleus

– Extraction of isolated centrosomes with 1 M potassium Extraction of isolated centrosomes with 1 M potassium iodide removes ~90% of PCM protein leaving behind iodide removes ~90% of PCM protein leaving behind spaghetti-like scaffold of insoluble fibersspaghetti-like scaffold of insoluble fibers

– Centrosomes are sites of convergence of large numbers Centrosomes are sites of convergence of large numbers of MTsof MTs

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


MT polymerization & disassembly - treat with MT polymerization & disassembly - treat with poisons (CO, NO) or cold —> MTs disassemble; poisons (CO, NO) or cold —> MTs disassemble; much has been learned about their disassembly & much has been learned about their disassembly & reassembly in cultured animal cells in this wayreassembly in cultured animal cells in this way– Observe assembly when cells warmed or poisons Observe assembly when cells warmed or poisons

removed; fix at various times after & stain with removed; fix at various times after & stain with fluorescent anti-tubulin ABsfluorescent anti-tubulin ABs

– Within a few minutes of inhibition removal, 1 or 2 Within a few minutes of inhibition removal, 1 or 2 bright fluorescent spots seen in cytoplasmbright fluorescent spots seen in cytoplasm

– Within 15 - 30 minutes, number of labeled filaments Within 15 - 30 minutes, number of labeled filaments radiating from these foci rises dramaticallyradiating from these foci rises dramatically

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 5. Microtubules : MTOCSC. 5. Microtubules : MTOCS– In EM: MTs radiate out from centrosome; MTs In EM: MTs radiate out from centrosome; MTs

don't actually penetrate into centrosome & don't actually penetrate into centrosome & contact centrioles, but terminate in dense contact centrioles, but terminate in dense pericentriolar material residing at centrosome pericentriolar material residing at centrosome peripheryperiphery

– PCM apparently initiates MT formation; PCM apparently initiates MT formation; centrioles not involved in MT nucleation, but centrioles not involved in MT nucleation, but they probably play a role in recruiting they probably play a role in recruiting surrounding PCM during centrosome assemblysurrounding PCM during centrosome assembly

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 5. MicrotubulesC. 5. Microtubules : MTOCS : MTOCS

Centrosome typically situated near center of cell, Centrosome typically situated near center of cell, just outside nucleus just outside nucleus – Columnar epithelium - centrosome moves from cell Columnar epithelium - centrosome moves from cell

center to apical region just beneath cortex; center to apical region just beneath cortex; cytoskeletal MTs emanate from site, extending toward cytoskeletal MTs emanate from site, extending toward nucleus & basal surface of cell nucleus & basal surface of cell

– Regardless of location, centrosomes are sites of MT Regardless of location, centrosomes are sites of MT nucleation; polarity is always the same: "-" end at nucleation; polarity is always the same: "-" end at centrosome, "+" (growing) end at opposite tipcentrosome, "+" (growing) end at opposite tip

– Thus, even though MTs are nucleated at MTOC, they Thus, even though MTs are nucleated at MTOC, they are elongated at opposite end of polymerare elongated at opposite end of polymer

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


Not all MTs associated with centrosome; Not all MTs associated with centrosome; – some animal cells (mouse oocytes) lack some animal cells (mouse oocytes) lack

centrosomes entirely, but still make spindlecentrosomes entirely, but still make spindle– MTs of axon are not associated with MTs of axon are not associated with

centrosome, which is located in cell body, but centrosome, which is located in cell body, but they may be formed at centrosome, then they may be formed at centrosome, then released from that MTOC & carried to axon released from that MTOC & carried to axon by motor proteinsby motor proteins

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


Basal bodies & other MTOCs Basal bodies & other MTOCs – Centrosomes are not the only MTOCs in cells; basal Centrosomes are not the only MTOCs in cells; basal

bodies at base of cilia & flagella serve as origin of bodies at base of cilia & flagella serve as origin of ciliary & flagellar MTs; MTs grow out of themciliary & flagellar MTs; MTs grow out of them

– Basal body cross-section looks like centriole; in fact, Basal body cross-section looks like centriole; in fact, the two can give rise to one another the two can give rise to one another

– Sperm flagellum arises from basal body derived from Sperm flagellum arises from basal body derived from sperm centriole that had been part of meiotic spindle sperm centriole that had been part of meiotic spindle of spermatocyte from which the sperm aroseof spermatocyte from which the sperm arose

– Conversely, sperm basal body typically becomes Conversely, sperm basal body typically becomes centriole during fertilized egg's first mitotic division centriole during fertilized egg's first mitotic division of fertilized eggof fertilized egg

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


Plant MTOC - lack both centrioles & Plant MTOC - lack both centrioles & centrosomes; MTOCs more dispersed than centrosomes; MTOCs more dispersed than those of animalsthose of animals– In plant endosperm cells, the primary MTOC In plant endosperm cells, the primary MTOC

consists of patches of material situated at outer consists of patches of material situated at outer surface of nuclear envelope from which surface of nuclear envelope from which cytoskeletal MTs emergecytoskeletal MTs emerge

– MT nucleation also thought to occur MT nucleation also thought to occur throughout plant cell cortexthroughout plant cell cortex

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


MT nucleationMT nucleation– Despite diverse appearances, all MTOCs play Despite diverse appearances, all MTOCs play

similar roles in all cellssimilar roles in all cells– Control number of MTs that form & their Control number of MTs that form & their

polaritypolarity– Control the number of protofilaments that Control the number of protofilaments that

make up their wallsmake up their walls– Control the time & location of MT assembly Control the time & location of MT assembly

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 5. Microtubules : MTOCSC. 5. Microtubules : MTOCS– All MTOCs share a common protein component, All MTOCs share a common protein component, --

tubulin (discovered in mid-1980s); it is ~0.005% of tubulin (discovered in mid-1980s); it is ~0.005% of total cell protein while total cell protein while - & - & -tubulins are 2.5% of -tubulins are 2.5% of total nonneural cell proteintotal nonneural cell protein

– Fluorescent anti-Fluorescent anti--tubulin antibodies (ABs) stain all -tubulin antibodies (ABs) stain all MTOCs, like centrosome PCM; suggests it is critical MTOCs, like centrosome PCM; suggests it is critical component in MT assembly & nucleationcomponent in MT assembly & nucleation

– Microinject anti-Microinject anti--tubulin AB into living cell —> -tubulin AB into living cell —> blocks MT reassembly after depolymerization by blocks MT reassembly after depolymerization by drugs or cold temperaturesdrugs or cold temperatures

– Genetically engineered fungi lacking functional g-Genetically engineered fungi lacking functional g-tubulin gene cannot assemble normal MTstubulin gene cannot assemble normal MTs

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 5. Microtubules : MTOCSC. 5. Microtubules : MTOCS Nucleation mechanism revealed by Nucleation mechanism revealed by

structure/composition studies of PCM at structure/composition studies of PCM at centrosome peripherycentrosome periphery– Insoluble fibers of PCM are thought to serve as Insoluble fibers of PCM are thought to serve as

attachment sites for ring-shaped structures that have attachment sites for ring-shaped structures that have same diameter as MTs (25 nm) & containsame diameter as MTs (25 nm) & contain-tubulin-tubulin

– Ring-shaped structures found when centrosomes were Ring-shaped structures found when centrosomes were purified & incubated with gold-labeled anti-purified & incubated with gold-labeled anti--tubulin -tubulin ABs —> cluster in rings/semi-circles at MT minus ends ABs —> cluster in rings/semi-circles at MT minus ends (ends embedded in PCM)(ends embedded in PCM)

– Isolate similar ring-shaped complexes (Isolate similar ring-shaped complexes (-TuRCs) from -TuRCs) from cell extracts; nucleate MT assembly in vitrocell extracts; nucleate MT assembly in vitro

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


Model - helical array of 13 Model - helical array of 13 -tubulin subunits -tubulin subunits forms open, ring-shaped template on which first forms open, ring-shaped template on which first row of row of -tubulin dimers assemble;-tubulin dimers assemble;– Only Only -tubulin of heterodimer can bind to ring of -tubulin of heterodimer can bind to ring of --

subunits, establishing polarity of entire MTsubunits, establishing polarity of entire MT

2 other tubulin isoforms 2 other tubulin isoforms -tubulin & -tubulin & -tubulin -tubulin have also been identified in centrosomes, but their have also been identified in centrosomes, but their function has not been determinedfunction has not been determined

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 6. Microtubules : DynamicC. 6. Microtubules : Dynamic

6.6. Dynamic properties of microtubulesDynamic properties of microtubules– Alberts: Table 16-2; Fig 16-16, 16-17Alberts: Table 16-2; Fig 16-16, 16-17– MTs vary markedly in stability even though similar MTs vary markedly in stability even though similar

morphologically - spindle/cytoskeleton labile; mature morphologically - spindle/cytoskeleton labile; mature neuron MTs less labile; cilia/flagella very stable; neuron MTs less labile; cilia/flagella very stable; lability allows cell to respond to stimulilability allows cell to respond to stimuli

– Cilia/flagella MTs are stabilized by MAP attachment Cilia/flagella MTs are stabilized by MAP attachment & by enzymatic modification (e. g. acetylation) of & by enzymatic modification (e. g. acetylation) of specific amino acid residues within tubulin subunitsspecific amino acid residues within tubulin subunits

– Labile MTs in living cells can be disassembled Labile MTs in living cells can be disassembled without disrupting other cell structures via a number without disrupting other cell structures via a number of treatmentsof treatments

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


Treatments that cause MT disassembly; Treatments that cause MT disassembly; usually interfere with noncovalent bonds usually interfere with noncovalent bonds holding them togetherholding them together– Cold temperaturesCold temperatures– Hydrostatic pressureHydrostatic pressure– Elevated CaElevated Ca2+2+ concentration concentration– Variety of chemicals (often used in Variety of chemicals (often used in

chemotherapy) - CO, vinblastine, vincristine, chemotherapy) - CO, vinblastine, vincristine, NO, podophyllotoxin NO, podophyllotoxin

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


Some treatments (taxol) disrupt MT Some treatments (taxol) disrupt MT dynamic activity & act by doing the dynamic activity & act by doing the opposite; inhibit disassemblyopposite; inhibit disassembly– Taxol binds MT polymer & thus prevents Taxol binds MT polymer & thus prevents

disassembly; cell cannot build new MT disassembly; cell cannot build new MT structuresstructures

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


Cytoskeletal MT lability reflects fact that they are Cytoskeletal MT lability reflects fact that they are polymers formed by noncovalent association of polymers formed by noncovalent association of dimers; subject to dimers; subject to depolymerization/repolymerization as cell needs depolymerization/repolymerization as cell needs changechange

Dramatic changes in MT spatial organization may Dramatic changes in MT spatial organization may be achieved by combination of 2 separate be achieved by combination of 2 separate mechanismsmechanisms– Rearrangement of existing MTsRearrangement of existing MTs– Disassembly of existing MTs & reassembly of new Disassembly of existing MTs & reassembly of new

ones in different cell regionsones in different cell regions

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


Study of MT dynamics in vitro - suggest that Study of MT dynamics in vitro - suggest that cytoskeleton can rapidly remodel & respond to cytoskeleton can rapidly remodel & respond to stimulistimuli– Early studies established that GTP binding to Early studies established that GTP binding to -subunit -subunit

required for MT assembly; GTP hydrolysis not needed required for MT assembly; GTP hydrolysis not needed for binding, but it is hydrolyzed soon after dimer attached for binding, but it is hydrolyzed soon after dimer attached to MT end; GDP stays boundto MT end; GDP stays bound

– After dimer is released from MT during disassembly & After dimer is released from MT during disassembly & enters soluble pool, GDP is replaced by GTP, thus enters soluble pool, GDP is replaced by GTP, thus recharging dimer so that it can add to polymer againrecharging dimer so that it can add to polymer again

– A GTP molecule is also bound to A GTP molecule is also bound to -tubulin subunit, but it -tubulin subunit, but it is not exchangeable & it is not hydrolyzed after subunit is not exchangeable & it is not hydrolyzed after subunit incorporationincorporation

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


Assembly is not energetically inexpensive Assembly is not energetically inexpensive since it includes GTP hydrolysis, but it does since it includes GTP hydrolysis, but it does allow the cell to control assembly & allow the cell to control assembly & disassembly independentlydisassembly independently– A dimer being added to MT has a bound GTP; A dimer being added to MT has a bound GTP;

dimer being released from MT has bound GDPdimer being released from MT has bound GDP– GDP- & GTP dimers have different GDP- & GTP dimers have different

conformations & participate in different conformations & participate in different reactions; the ends of growing & shrinking reactions; the ends of growing & shrinking MTs have different structures MTs have different structures

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


The above facts lead to the following model:The above facts lead to the following model:– When a MT is growing, the "+" end is present as an When a MT is growing, the "+" end is present as an

open sheet to which GTP-dimers are addedopen sheet to which GTP-dimers are added

– During rapid growth periods, tubulin dimers are added During rapid growth periods, tubulin dimers are added faster than GTP can be hydrolyzedfaster than GTP can be hydrolyzed

– The resultant cap of GTP-dimers on MT at The resultant cap of GTP-dimers on MT at protofilament ends is thought to favor the addition of protofilament ends is thought to favor the addition of more subunits & hence MT growthmore subunits & hence MT growth

– However, MTs with open ends thought to undergo However, MTs with open ends thought to undergo spontaneous reaction leading to tube closurespontaneous reaction leading to tube closure

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


– tube closure is accompanied by hydrolysis of tube closure is accompanied by hydrolysis of bound GTP, changing tubulin dimer bound GTP, changing tubulin dimer conformation —> resultant mechanical strain conformation —> resultant mechanical strain destabilizes MTsdestabilizes MTs

– Strain is released as protofilaments curl out Strain is released as protofilaments curl out from tubule & catastrophically depolymerizefrom tubule & catastrophically depolymerize

– Disassembly can occur remarkably fast, Disassembly can occur remarkably fast, especially in vivo, which allows very rapid MT especially in vivo, which allows very rapid MT cytoskeleton disassemblycytoskeleton disassembly

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


Study of MT dynamics in vivo: dynamic character of Study of MT dynamics in vivo: dynamic character of MT cytoskeleton inside cell is best revealed by MT cytoskeleton inside cell is best revealed by microinjecting labeled tubulin into nondividing microinjecting labeled tubulin into nondividing cultured cellcultured cell– Inject labeled tubulin into nondividing cultured cell —> Inject labeled tubulin into nondividing cultured cell —>

labeled subunits rapidly incorporated into preexisting labeled subunits rapidly incorporated into preexisting cytoskeleton MTs, even in absence of any obvious cytoskeleton MTs, even in absence of any obvious morphological changemorphological change

– Watch cell with fluorescent-labeled MTs over time —> Watch cell with fluorescent-labeled MTs over time —> some MTs grow, others shrink; dynamicsome MTs grow, others shrink; dynamic

– Both growth & shrinkage in vivo occur predominantly at Both growth & shrinkage in vivo occur predominantly at "+" end of polymer, the end located opposite the "+" end of polymer, the end located opposite the centrosome (or other MTOC)centrosome (or other MTOC)

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments


– Single MTs switch randomly & unpredictably Single MTs switch randomly & unpredictably between growing & shrinking (dynamic between growing & shrinking (dynamic instability)instability)

– MTs shrink faster than they grow, so in a MTs shrink faster than they grow, so in a matter of minutes, MTs disappear & are matter of minutes, MTs disappear & are replaced by new MTs that grow out from replaced by new MTs that grow out from centrosomecentrosome

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 7. Microtubules: FlagellaC. 7. Microtubules: Flagella

7.7. Cilia and flagella structureCilia and flagella structure– Alberts: Fig 16-80, 81, 82, 83, 84Alberts: Fig 16-80, 81, 82, 83, 84

Entire ciliary or flagellar projection is covered Entire ciliary or flagellar projection is covered by membrane continuous with cell membraneby membrane continuous with cell membrane

Cilium core (axoneme) contains an array of MTs Cilium core (axoneme) contains an array of MTs that run longitudinally through entire organelle that run longitudinally through entire organelle

– Usually 9 peripheral doublet MTs surrounding central Usually 9 peripheral doublet MTs surrounding central pair of single MTs; known as 9 + 2 pattern or array; pair of single MTs; known as 9 + 2 pattern or array; all MTs in array have same polarity ("+" ends at tip, all MTs in array have same polarity ("+" ends at tip, "-" ends at base)"-" ends at base)

– Doublets - 1 complete (A tubule; 13 subunits) MT; 1 Doublets - 1 complete (A tubule; 13 subunits) MT; 1 incomplete (B tubule) MT with 10 or 11 subunitsincomplete (B tubule) MT with 10 or 11 subunits

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 7. Microtubules: FlagellaC. 7. Microtubules: Flagella– Not all eukaryotes have them; cilia & flagella generally Not all eukaryotes have them; cilia & flagella generally

absent among fungi, nematodes & insectsabsent among fungi, nematodes & insects

– Where they do occur, they nearly always show same 9 Where they do occur, they nearly always show same 9 + 2 array, a reminder that all living eukaryotes have + 2 array, a reminder that all living eukaryotes have evolved from a common ancestorevolved from a common ancestor

– Despite high degree of conservation (e. g. 9 + 2 pattern) Despite high degree of conservation (e. g. 9 + 2 pattern) some evolutionary departures: some evolutionary departures:

» 9 + 1 array in flatworms9 + 1 array in flatworms

» 9 + 0 array in Asian horseshoe crab, eel, mayfly; some lacking 9 + 0 array in Asian horseshoe crab, eel, mayfly; some lacking central elements are motile, some notcentral elements are motile, some not

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 7. Microtubules: FlagellaC. 7. Microtubules: Flagella Central MTs enclosed by projections forming Central MTs enclosed by projections forming

central sheath; sheath connected to doublet A MTs central sheath; sheath connected to doublet A MTs by radial spokes; doublets connected by by radial spokes; doublets connected by interdoublet bridge made of elastic protein nexininterdoublet bridge made of elastic protein nexin

Pair of arms (inner & outer) project from A MT in Pair of arms (inner & outer) project from A MT in clockwise direction (when viewed base to tip)clockwise direction (when viewed base to tip)

Radial spokes typically in groups of three with Radial spokes typically in groups of three with major repeat of 96 nmmajor repeat of 96 nm

Inner & outer dynein arms staggered along A MT Inner & outer dynein arms staggered along A MT length (outer arms spaced every 24 nm; inner arms length (outer arms spaced every 24 nm; inner arms arranged to match unequal spacing of radial spokes)arranged to match unequal spacing of radial spokes)

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 7. Microtubules: FlagellaC. 7. Microtubules: Flagella Cilia/flagellae emerge from basal bodies - 9 Cilia/flagellae emerge from basal bodies - 9

peripheral fibers consisting of 3 MTs rather peripheral fibers consisting of 3 MTs rather than 2 (A tube complete, B/C incomplete); than 2 (A tube complete, B/C incomplete); similar in structure to centriolessimilar in structure to centrioles– No central MTs as in centrioles; also similar to No central MTs as in centrioles; also similar to

centrioles in other wayscentrioles in other ways– A & B tubules elongate to form cilia/flagella A & B tubules elongate to form cilia/flagella

doublet; if sheared off, regrow from basal bodydoublet; if sheared off, regrow from basal body

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 7. Microtubules: FlagellaC. 7. Microtubules: Flagella The mechanism of ciliary & flagellar locomotion: The mechanism of ciliary & flagellar locomotion:

sliding filament model suggested mechanism of sliding filament model suggested mechanism of ciliary/flagellar movement was sliding of adjacent ciliary/flagellar movement was sliding of adjacent MT doublets relative to one anotherMT doublets relative to one another

In model, dynein arms act as swinging cross-In model, dynein arms act as swinging cross-bridges that generate forces needed for bridges that generate forces needed for ciliary/flagellar movement; dynein arms projecting ciliary/flagellar movement; dynein arms projecting from one doublet walk along adjacent doublet wall from one doublet walk along adjacent doublet wall —> sliding—> sliding

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

C. 7. Microtubules: FlagellaC. 7. Microtubules: Flagella Sequence of events in ciliary/flagellar sliding Sequence of events in ciliary/flagellar sliding

motionmotion– Dynein arms anchored on a doublet's A MT attach to Dynein arms anchored on a doublet's A MT attach to

binding sites on B MT of adjacent doubletbinding sites on B MT of adjacent doublet

– Dynein molecules undergo conformational change; Dynein molecules undergo conformational change; causes A MT doublet to move slightly toward basal end causes A MT doublet to move slightly toward basal end of attached B MT doubletof attached B MT doublet

– Dynein then releases B tubule of adjacent doubletDynein then releases B tubule of adjacent doublet

– Dynein arms reattach to adjacent doublet's B MT closer Dynein arms reattach to adjacent doublet's B MT closer to its base so another cycle can beginto its base so another cycle can begin

A. Overview


C. Microtubules

1. Structure

2. MAPs

3. Functions


5. MTOCs



D. Microfilaments

D.D. MicrofilamentsMicrofilaments1.1. StructureStructure

2.2. Polymerization/depolymerizationPolymerization/depolymerization

3.3. MyosinMyosin

4.4. Muscle ContractionMuscle Contraction

5.5. Non-muscle motilityNon-muscle motility

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins

4. Muscle Contraction

5. Nonmuscle Actin

D. 1. Microfilaments: StructureD. 1. Microfilaments: Structure

1.1. StructureStructure Alberts Fig 16-12Alberts Fig 16-12 Microfilaments:Microfilaments:

– ~8 nm diameter~8 nm diameter– made of globular actin subunits (G-actin) made of globular actin subunits (G-actin) – found in most animal cells, also higher plantsfound in most animal cells, also higher plants– ““Microfilament,” “actin filament,” “F-actin Microfilament,” “actin filament,” “F-actin

filaments” are synonyms but F-actin often filaments” are synonyms but F-actin often used for those formed in vitroused for those formed in vitro

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


In presence of ATP, G-actin polymerizes to form In presence of ATP, G-actin polymerizes to form stiff filament made of 2 strands of F-actin wound stiff filament made of 2 strands of F-actin wound around each other in a helical configuration around each other in a helical configuration

– Each subunit has polarity & all subunits are pointed Each subunit has polarity & all subunits are pointed in same direction, so entire MF has polarityin same direction, so entire MF has polarity

Depending on cell type & activity in which it is Depending on cell type & activity in which it is engaged, MFs can be organized into highly engaged, MFs can be organized into highly ordered arrays, loose ill-defined networks, or ordered arrays, loose ill-defined networks, or tightly anchored bundles tightly anchored bundles

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


Actin identified more than 50 years ago as one of Actin identified more than 50 years ago as one of major contractile proteins of muscle cellsmajor contractile proteins of muscle cells– Since then found to be major protein in virtually every Since then found to be major protein in virtually every

eukaryotic cell examinedeukaryotic cell examined

– Higher plants & animals possess number of actin-Higher plants & animals possess number of actin-coding genes whose products are specialized for coding genes whose products are specialized for different types of motile processesdifferent types of motile processes

– Actin structure highly conserved evolutionarily (yeast Actin structure highly conserved evolutionarily (yeast actin & rabbit skeletal actin 88% identical); means that actin & rabbit skeletal actin 88% identical); means that nearly all aminos are crucial to function; actin from nearly all aminos are crucial to function; actin from diverse sources can copolymerizediverse sources can copolymerize

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


Actin detected microscopically: Actin detected microscopically: – By electron microscopy, using proteolytically cleaved By electron microscopy, using proteolytically cleaved

myosin head fragments (HMM or S1 fragments)myosin head fragments (HMM or S1 fragments)» HMM & S1 bind actin all along MF —> see polarity in EM; HMM & S1 bind actin all along MF —> see polarity in EM;

one end of MF pointed, other end barbedone end of MF pointed, other end barbed

» Orientation of arrowheads formed by S1-actin complex Orientation of arrowheads formed by S1-actin complex provides information as to direction in which MFs are likely to provides information as to direction in which MFs are likely to be moved by myosin motor proteinbe moved by myosin motor protein

– By fluorescence microscopy, with fluorescently labeled By fluorescence microscopy, with fluorescently labeled S1 or anti-actin ABsS1 or anti-actin ABs

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin

D. 2. Microfilaments: P/DD. 2. Microfilaments: P/D2.2. MF polymerization/depolymerizationMF polymerization/depolymerization Alberts Table 16 – 2, Fig. 16 – 36, 37, 38 Alberts Table 16 – 2, Fig. 16 – 36, 37, 38 Before polymerization, actin monomer binds to Before polymerization, actin monomer binds to

adenosine nucleotide (usually ATP); like GTP in adenosine nucleotide (usually ATP); like GTP in MTsMTs

– Actin is an ATPase (like tubulin is GTPase); role of Actin is an ATPase (like tubulin is GTPase); role of ATP in MF assembly is same as GTP in MTATP in MF assembly is same as GTP in MT

– Some time after incorporation into growing actin Some time after incorporation into growing actin filament, ATP hydrolyzed to ADPfilament, ATP hydrolyzed to ADP

If filaments built at high rate, the end has actin-If filaments built at high rate, the end has actin-ATP cap (hinders disassembly, favors assembly)ATP cap (hinders disassembly, favors assembly)

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin

D. 2. Microfilaments: P/DD. 2. Microfilaments: P/D Actin polymerization can be studied in vitro by Actin polymerization can be studied in vitro by

labeling or viscosity studieslabeling or viscosity studies– In vitro with high concentration of labeled G-actin, both In vitro with high concentration of labeled G-actin, both

ends labeled but .....ends labeled but .....» One end incorporates monomers at 5 - 10 times higher rate than the One end incorporates monomers at 5 - 10 times higher rate than the

other endother end» Decoration with S1 myosin fragment reveals that barbed ("+" end) Decoration with S1 myosin fragment reveals that barbed ("+" end)

of MF is fast-growing end, while the pointed ("-") end is the slow-of MF is fast-growing end, while the pointed ("-") end is the slow-growing tipgrowing tip

– In lower concentrations of G-actin:In lower concentrations of G-actin:» Actin-ATP subunits add to "+" end & actin-ADP subunits tend to Actin-ATP subunits add to "+" end & actin-ADP subunits tend to

leave from "-" leave from "-" » Can be demonstrated by pulse-chase “treadmilling” experimentsCan be demonstrated by pulse-chase “treadmilling” experiments» Don’t know if treadmilling occurs in vivoDon’t know if treadmilling occurs in vivo

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin

D. 2. Microfilaments: P/DD. 2. Microfilaments: P/D MFs maintain a dynamic equilibrium between MFs maintain a dynamic equilibrium between

monomeric & polymeric actin - can be influenced monomeric & polymeric actin - can be influenced by a variety of different proteinsby a variety of different proteins

Changes in local conditions in particular part of Changes in local conditions in particular part of cell can push equilibrium either toward assembly cell can push equilibrium either toward assembly or disassemblyor disassembly– allows cell to reorganize its MFs cytoskeleton by allows cell to reorganize its MFs cytoskeleton by

controlling this equilibriumcontrolling this equilibrium

– need such reorganization for dynamic processes (cell need such reorganization for dynamic processes (cell locomotion & shape changes, cytokinesis)locomotion & shape changes, cytokinesis)

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin

D. 2. Microfilaments: P/DD. 2. Microfilaments: P/D Actin-binding proteins in the cell affect the nucleation and Actin-binding proteins in the cell affect the nucleation and

polymerization rate of microfilamentspolymerization rate of microfilaments– Formin: A dimeric protein that initiates nucleation by capturing Formin: A dimeric protein that initiates nucleation by capturing

two monomers of actin, then remains associated with the plus end two monomers of actin, then remains associated with the plus end of a rapidly growing microfolamentof a rapidly growing microfolament

– Thymosin: A protein that binds to actin monomers and inhibits Thymosin: A protein that binds to actin monomers and inhibits nucleotide exchange or polymerization, keeping much of the nucleotide exchange or polymerization, keeping much of the available actin in an unpolymerized stateavailable actin in an unpolymerized state

– Profilin: A protien that competes with thymosin for binding to Profilin: A protien that competes with thymosin for binding to actin monomers; it binds opposite the ATP binding site on actin actin monomers; it binds opposite the ATP binding site on actin and promotes polymerization at the plus end of a growing filament and promotes polymerization at the plus end of a growing filament

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin

D. 2. Microfilaments: P/DD. 2. Microfilaments: P/D Inhibitors of microfilament Inhibitors of microfilament

polymerization/depolymerization used to polymerization/depolymerization used to study microfilament polymerization (Table study microfilament polymerization (Table 16 - 216 - 2

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin

D. 3. Microfilaments: MyosinsD. 3. Microfilaments: Myosins

3.3. MyosinsMyosins Alberts fig 16 – 54, 55, 56, 57, 60, 61, 65, 68, 69, 72Alberts fig 16 – 54, 55, 56, 57, 60, 61, 65, 68, 69, 72 Myosin's sole known function is as motor for actin; Myosin's sole known function is as motor for actin;

– Almost all motors known to interact with actin are members of Almost all motors known to interact with actin are members of myosin superfamilymyosin superfamily

– all of them move toward MF plus end (except for myosin VI)all of them move toward MF plus end (except for myosin VI)– First isolated from mammalian skeletal muscle & then from First isolated from mammalian skeletal muscle & then from

wide variety of eukaryotic cells: protists, higher plants, wide variety of eukaryotic cells: protists, higher plants, nonmuscle animal cells, vertebrate cardiac & smooth muscle nonmuscle animal cells, vertebrate cardiac & smooth muscle tissuestissues

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


Structure of myosinsStructure of myosins– All share characteristic motor (head) domain, which All share characteristic motor (head) domain, which

has a site that binds actin filament & one that binds & has a site that binds actin filament & one that binds & hydrolyzes ATP to drive the myosin motorhydrolyzes ATP to drive the myosin motor

– While head domains of myosins are similar, tail While head domains of myosins are similar, tail domains are highly divergentdomains are highly divergent

– Myosins also contain variety of low molecular weight Myosins also contain variety of low molecular weight (light) chains(light) chains

– Based on these construction differences divided into 2 Based on these construction differences divided into 2 large groups - conventional & unconventionallarge groups - conventional & unconventional

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


Conventional (type II): Conventional (type II): – found in various muscle tissues, and also in a variety of found in various muscle tissues, and also in a variety of

nonmuscle cells (generate tension at focal contacts, nonmuscle cells (generate tension at focal contacts, cytokinesis)cytokinesis)

– Structure of myosin II molecules: 6 polypeptide chains (one Structure of myosin II molecules: 6 polypeptide chains (one pair of heavy chains, 2 pairs of light chains); organized in a pair of heavy chains, 2 pairs of light chains); organized in a way that produces a highly asymmetric protein with 3 way that produces a highly asymmetric protein with 3 sectionssections

» A pair of globular heads that contain the molecule’s catalytic siteA pair of globular heads that contain the molecule’s catalytic site» A pair of necks, each consisting of a single, uninterrupted a-helix & A pair of necks, each consisting of a single, uninterrupted a-helix &

2 associated light chains2 associated light chains» A single, long, rod-shaped tail formed by the intertwining of long A single, long, rod-shaped tail formed by the intertwining of long

a-helical sections of the 2 heavy chains to form an a-helical coiled-a-helical sections of the 2 heavy chains to form an a-helical coiled-coilcoil

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


Immobilize isolated myosin heads (S1 fragments) Immobilize isolated myosin heads (S1 fragments) on glass cover slip —> cause actin filament on glass cover slip —> cause actin filament slidingsliding– Single head domain has all of the machinery needed for Single head domain has all of the machinery needed for

motor activitymotor activity The fibrous tail plays a structural role, allowing The fibrous tail plays a structural role, allowing

the protein to form filamentsthe protein to form filaments Light chain phosphorylation regulates assembly of Light chain phosphorylation regulates assembly of

myosin II into thick filamentsmyosin II into thick filaments Tail ends of myosin molecule point toward Tail ends of myosin molecule point toward

filament center; heads point toward ends (bipolar)filament center; heads point toward ends (bipolar)– Polarity of filament reverses at its centerPolarity of filament reverses at its center

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


Skeletal muscle myosin II filaments are Skeletal muscle myosin II filaments are highly stablehighly stable

smaller myosin II filaments (most smaller myosin II filaments (most nonmuscle cells) often display transient nonmuscle cells) often display transient construction (assembling when & where construction (assembling when & where needed, then disassembling)needed, then disassembling)

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


Unconventional myosins subdivided into at least Unconventional myosins subdivided into at least 14 different types 14 different types – Each type is presumed to have its own specialized Each type is presumed to have its own specialized

functionsfunctions– Several types may be present together in same cellSeveral types may be present together in same cell

Some functions of unconventional myosinsSome functions of unconventional myosins– Amoeboid movement & phagocytosis (myosin I)Amoeboid movement & phagocytosis (myosin I)– Movement of cytoplasmic vesicles & organelles Movement of cytoplasmic vesicles & organelles

(myosins I, V, & VI)(myosins I, V, & VI)– Stereocilia in cochlea hair cells of inner ear (myosin Stereocilia in cochlea hair cells of inner ear (myosin

VIIa)VIIa)

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin

D. 4. Microfilaments: MuscleD. 4. Microfilaments: Muscle

4.4. Muscle contractionMuscle contraction Alberts 16 – 73, 74, 75, 76, 61, 77, 78Alberts 16 – 73, 74, 75, 76, 61, 77, 78 Skeletal muscle cell structure - highly Skeletal muscle cell structure - highly

unorthodox; cylindrical; 10 - 100 µm thick; up unorthodox; cylindrical; 10 - 100 µm thick; up to 400 mm longto 400 mm long

– Skeletal muscle cells are multinucleate (100s), the Skeletal muscle cells are multinucleate (100s), the result of embryonic fusion of mononucleate result of embryonic fusion of mononucleate myoblasts (premuscle cells); even myoblasts from myoblasts (premuscle cells); even myoblasts from distantly related animals fuse in culturedistantly related animals fuse in culture

– Because of their properties, these cells are more Because of their properties, these cells are more appropriately called muscle fibersappropriately called muscle fibers

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


Muscle fibers may have the most orderly internal Muscle fibers may have the most orderly internal structure of any cell in bodystructure of any cell in body– Muscle fiber is cable made up of hundreds of thinner, Muscle fiber is cable made up of hundreds of thinner,

cylindrical strands (myofibrils)cylindrical strands (myofibrils)– Each myofibril is repeating linear array of contractile Each myofibril is repeating linear array of contractile

units (sarcomeres) units (sarcomeres) – Each sarcomere, in turn, has characteristic banding Each sarcomere, in turn, has characteristic banding

pattern that gives muscle fiber a striated lookpattern that gives muscle fiber a striated look Myofibrils separated by cytoplasm with Myofibrils separated by cytoplasm with

intracellular membranes & mitochondria, lipid intracellular membranes & mitochondria, lipid droplets, glycogen granulesdroplets, glycogen granules

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


Banding pattern is result of partial overlap Banding pattern is result of partial overlap between thick & thin filamentsbetween thick & thin filaments– Each sarcomere extends from Z line to Z line (~2.5 µm) Each sarcomere extends from Z line to Z line (~2.5 µm)

& contains several dark bands & light zones; there is a & contains several dark bands & light zones; there is a pair of light staining I bands at each end of sarcomerepair of light staining I bands at each end of sarcomere

– More densely staining A band is between outer I bands; More densely staining A band is between outer I bands; lightly staining H zone in A band centerlightly staining H zone in A band center

– Densely staining M line lies in center of H zoneDensely staining M line lies in center of H zone

– I bands - only thin filaments; H zone - only thick I bands - only thin filaments; H zone - only thick filaments; A band outside H zone - both overlapfilaments; A band outside H zone - both overlap

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


Composition & organization of thin filamentsComposition & organization of thin filaments– Thin filaments mostly actinThin filaments mostly actin– In addition to actin, thin filaments also contain two In addition to actin, thin filaments also contain two

other proteins: troponin & tropomyosinother proteins: troponin & tropomyosin» Tropomyosin - elongated, ~40 nm long; fits securely into Tropomyosin - elongated, ~40 nm long; fits securely into

grooves between two thin filament actin chains; each rod-grooves between two thin filament actin chains; each rod-shaped tropomyosin interacts with 7 actin subunits linearly shaped tropomyosin interacts with 7 actin subunits linearly along F-actin chainalong F-actin chain

» Troponin - globular protein complex with 3 subunits - each has Troponin - globular protein complex with 3 subunits - each has distinct, important functional role; ~40 nm apart on thin distinct, important functional role; ~40 nm apart on thin filament, contact both actin & tropomyosin thin filament filament, contact both actin & tropomyosin thin filament componentscomponents

– Actin filaments of each half sarcomere aligned with Actin filaments of each half sarcomere aligned with barbed ends linked to Z line by barbed ends linked to Z line by -actinin-actinin

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


Composition & organization of thick filamentsComposition & organization of thick filaments– Thick filaments are several 100 myosins & a small Thick filaments are several 100 myosins & a small

number of other proteins number of other proteins

– Like filaments formed in vitro, thick filament polarity Like filaments formed in vitro, thick filament polarity is reversed at sarcomere center; filament center is is reversed at sarcomere center; filament center is composed of opposing tail regions of myosin molecules composed of opposing tail regions of myosin molecules & is devoid of heads & is devoid of heads

– Myosin heads project from each thick filament along Myosin heads project from each thick filament along remainder of its length due to staggered positions of remainder of its length due to staggered positions of myosins making up the body of the filamentmyosins making up the body of the filament

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


– Titin (~3 x 106 dalton MW [27,000 aa’s], 1 µm Titin (~3 x 106 dalton MW [27,000 aa’s], 1 µm long) - largest protein yet discovered; originates long) - largest protein yet discovered; originates at M line & extends along myosin filament past at M line & extends along myosin filament past A band to terminate on Z lineA band to terminate on Z line

» Highly elastic protein; stretches (bungee cord) as Highly elastic protein; stretches (bungee cord) as certain domains within molecule are unfolded certain domains within molecule are unfolded

» It is thought to prevent sarcomere from being pulled It is thought to prevent sarcomere from being pulled apart during muscle stretchingapart during muscle stretching

» Also maintains myosin filaments in proper position Also maintains myosin filaments in proper position in sarcomere center during contractionin sarcomere center during contraction

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin

The Sliding Filament Model of muscle contractionThe Sliding Filament Model of muscle contraction– muscles contract by shortening at sarcomere levelmuscles contract by shortening at sarcomere level

– combined decrease in sarcomere length accounts for combined decrease in sarcomere length accounts for decrease in length of entire muscledecrease in length of entire muscle

– Sarcomere banding patterns shift during contraction - Sarcomere banding patterns shift during contraction - shows that filaments slide shows that filaments slide

» H zone & I bands decrease in width during contraction & H zone & I bands decrease in width during contraction & eventually disappear; A bands stay sameeventually disappear; A bands stay same

» During contraction, Z lines move inward & approach A band During contraction, Z lines move inward & approach A band outer edges until they touch each otherouter edges until they touch each other


A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


Molecular basis of contraction Molecular basis of contraction – During contraction, each myosin head extends out & During contraction, each myosin head extends out &

binds tightly to thin filaments forming cross-bridges binds tightly to thin filaments forming cross-bridges – Once bridges form, heads change shape, bending Once bridges form, heads change shape, bending

toward sarcomere center (power stroke)toward sarcomere center (power stroke)– Moves actin filament over thick filament (~5 - 15 nm; Moves actin filament over thick filament (~5 - 15 nm;

controversy exists over these distances) toward controversy exists over these distances) toward sarcomere center sarcomere center

– Heads hydrolyze ATP & act as levers for thin filament Heads hydrolyze ATP & act as levers for thin filament motion; each myosin cross-bridge mechanical activity motion; each myosin cross-bridge mechanical activity cycle is accompanied by ATPase activity cyclecycle is accompanied by ATPase activity cycle

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


Details of the sliding filament modelDetails of the sliding filament model– Cycle starts with ATP binding to myosin head, which Cycle starts with ATP binding to myosin head, which

induces dissociation of cross-bridge from actin induces dissociation of cross-bridge from actin – ATP binding followed by its hydrolysis before head ATP binding followed by its hydrolysis before head

contacts actin filament; ADP & Pi products stay bound contacts actin filament; ADP & Pi products stay bound at enzyme active siteat enzyme active site

– Energy released by hydrolysis absorbed by myosin as a Energy released by hydrolysis absorbed by myosin as a whole, placing the cross-bridge in energized state, like whole, placing the cross-bridge in energized state, like a stretched spring capable of spontaneous movementa stretched spring capable of spontaneous movement

– Energized myosin attaches to actin & releases its bound Energized myosin attaches to actin & releases its bound PiPi

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


– Triggers a large conformational change, driven by Triggers a large conformational change, driven by stored free energystored free energy

– The shape change shifts actin filament toward The shape change shifts actin filament toward sarcomere center (myosin head power stroke)sarcomere center (myosin head power stroke)

– Bound ADP leaves & new ATP attaches, inducing Bound ADP leaves & new ATP attaches, inducing release of cross-bridge; cycle starts overrelease of cross-bridge; cycle starts over

– In absence of ATP, cross-bridges stay tightly intact In absence of ATP, cross-bridges stay tightly intact binding actin (causes rigor mortis after death)binding actin (causes rigor mortis after death)

Skeletal muscle contraction is triggered by a Skeletal muscle contraction is triggered by a nerve-impulse mediated release of calcium ions nerve-impulse mediated release of calcium ions into the sarcomereinto the sarcomere

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


Regulation of skeletal muscle contractionRegulation of skeletal muscle contraction– Muscle fibers are organized into motor units; Muscle fibers are organized into motor units;

all fibers of motor unit jointly innervated by all fibers of motor unit jointly innervated by branches of one motor neuron; contract branches of one motor neuron; contract simultaneously if stimulated by impulse simultaneously if stimulated by impulse transmitted along that neurontransmitted along that neuron

» Point where neuron axon terminus & muscle fiber Point where neuron axon terminus & muscle fiber make contact called neuromuscular junctionmake contact called neuromuscular junction

» Junction is site of nerve impulse transmission from Junction is site of nerve impulse transmission from axon across synaptic cleft to muscle fiberaxon across synaptic cleft to muscle fiber

» Muscle fiber plasma membrane is also excitable & Muscle fiber plasma membrane is also excitable & capable of conducting an action potentialcapable of conducting an action potential

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


– Unlike a neuron, where an action potential stays Unlike a neuron, where an action potential stays at the cell surface, skeletal muscle cell impulse at the cell surface, skeletal muscle cell impulse is propagated into cell interior along is propagated into cell interior along membranous folds (transverse [T] tubules)membranous folds (transverse [T] tubules)

» T tubules terminate in close proximity to a T tubules terminate in close proximity to a cytoplasmic membrane system (sarcoplasmic cytoplasmic membrane system (sarcoplasmic reticulum [SR]) that forms membranous sleeve reticulum [SR]) that forms membranous sleeve around myofibril (specialized form of smooth ER)around myofibril (specialized form of smooth ER)

» ~80% of SR membrane integral protein is ATP-~80% of SR membrane integral protein is ATP-driven Cadriven Ca2+2+ pump (moves Ca pump (moves Ca2+2+ from cytosol into SR from cytosol into SR lumen where it is stored until it is needed)lumen where it is stored until it is needed)

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin

– With arrival of action potential at SR via T With arrival of action potential at SR via T tubules, opens Catubules, opens Ca2+2+ channels in SR membrane channels in SR membrane are openedare opened

» CaCa2+2+ diffuses out of SR compartment & over short diffuses out of SR compartment & over short distance to myofibrilsdistance to myofibrils

» [Ca[Ca+2+2] levels go from ~2 x 10] levels go from ~2 x 10-7-7 M to ~5 x 10 M to ~5 x 10-5-5 M M

– In a relaxed sarcomere (low [CaIn a relaxed sarcomere (low [Ca22+]):+]):» Tropomyosin blocks myosin-binding sites on actin Tropomyosin blocks myosin-binding sites on actin

moleculesmolecules

» Tropomyosin’s position in filament groove blocking Tropomyosin’s position in filament groove blocking sites is controlled by troponinsites is controlled by troponin

D. 4. Microfilaments: Muscle

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


– When [Ca2+] rises: When [Ca2+] rises: » CaCa2+2+ binds to troponin C subunits binds to troponin C subunits » This causes a conformational change in troponin This causes a conformational change in troponin » And this causes adjacent tropomyosin to move ~1.5 And this causes adjacent tropomyosin to move ~1.5

nm closer to center of filament’s groovenm closer to center of filament’s groove

– The movement of tropomyosin exposes The movement of tropomyosin exposes myosin-binding site on adjacent actins, cross-myosin-binding site on adjacent actins, cross-bridges form, contraction occurs bridges form, contraction occurs

– Each troponin controls position of 1 Each troponin controls position of 1 tropomyosin, which, in turn, controls binding tropomyosin, which, in turn, controls binding capacity of 7 adjacent actin monomerscapacity of 7 adjacent actin monomers

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin


– When stimulation from motor neuron stops:When stimulation from motor neuron stops:» SR CaSR Ca2+2+ channels close & excess Ca channels close & excess Ca2+2+ pumped from pumped from

cytosol back into SR by the Cacytosol back into SR by the Ca2+2+ pump pump

» As [CaAs [Ca2+2+] decreases, Ca] decreases, Ca2+2+ ions dissociate from ions dissociate from troponin binding sitestroponin binding sites

» Tropomyosin molecules return to their original Tropomyosin molecules return to their original positions and block actin-myosin bindingpositions and block actin-myosin binding

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin

D. 5. Microfilaments: NonmuscleD. 5. Microfilaments: Nonmuscle5.5. Some examples of nonmuscle actin-myosin Some examples of nonmuscle actin-myosin

– Alberts Fig. 16 – 50, 51, 52Alberts Fig. 16 – 50, 51, 52– CytokinesisCytokinesis– PhagocytosisPhagocytosis– Cytoplasmic streaming (directed bulk flow of cytoplasm Cytoplasmic streaming (directed bulk flow of cytoplasm

occurring in certain large plant cells)occurring in certain large plant cells)– Vesicle traffickingVesicle trafficking– Blood platelet activationBlood platelet activation– Lateral movements of integral proteins within membranesLateral movements of integral proteins within membranes– Cell substratum interactions, cell locomotion & axonal Cell substratum interactions, cell locomotion & axonal

outgrowthoutgrowth– Changes in cell shapeChanges in cell shape– Cell projections such as microvilli & stereociliaCell projections such as microvilli & stereocilia

A. Overview


C. Microtubules

D. Microfilaments

1. Structure

2. P/D

3. Myosins


5. Nonmuscle Actin

Documents

Cytoskeleton A. Overview B. Experimental Methods C. Microtubules D. Microfilaments (Updated 4/9/08) A. Overview B. Experimental Methods C. Microtubules