Cytomechanics432/532Tuesday, January 18, 2005: IntroductionWebCT syllabus, book, resources, posting.Office : BME 124 Weds, Thurs: 1-4 PMGrading: HW + Exams + ProjectCraelius@rci

Learning objectives1. To learn the structural/mechanical components of cells, specifically: biophysics and material properties of the cytoskeleton (CSK), membrane, and matrix. 2. To learn about experimental tools for evaluating cell mechanical properties, specifically: mechanical testing, imaging with immunocytochemisty and knock-out methods.

Learning Objectives3. To learn kinematics and dynamics of cells, specifically, interactions among CSK, cytosol, matrix, and nucleus, mechanotransduction, and motility4. To learn statistical mechanics of cell polymers and CSK assembly. 5. To learn tools for modelling cell mechanics, specifically simulations with matlab and simulink.

Topics in CytomechanicsA cell is a cytoplasmic structural element.Tensegrity holds it together -centripetally.Structural components include lipids, and 3 separate filament systems. No cell is an island- interactions with others and the ECM shape and regulate it.Trans-skeletal molecules regulate the cell.Rxns in solid-state versus enzyme solution.

QuestionsHow do cells maintain and change shape?Move?Grow and maintain a size?Anchor to substrate or stick together or not?Transport materials inside?Form tissues?Sense force and deformation?

Applications of Cytomechanics?MedicalStress-Growth HypothesisMechanoelectrical FeedbackTumor-EndotheliumWound HealingEdemaBone & Cartilage ControlCellular signalling

TechnologicalGas structural elementsMotility of GelsMicrotubular nanostructuresBioprocess optimizationPlant Growth & ProductionMicrogravity Effects

How are cells put together? 200 different typesNot nice and regularVaried and irregular

The generic cell

Tension + compression hold the cell together

Green fluorescent dye for Actin

Basic Cell Components A membrane, skeleton, and internal structures.All serve both as structural and functional elements.Simplified basic building blocks

Geodesic- Buckminster Fuller A geodesic dome uses a pattern of self-bracing triangles in a pattern that gives maximum structural advantage, thus theoretically using the least material possible. (A "geodesic" line on a sphere is the shortest distance between any two points.)

Stick Geodesic Domes : Ingber

Tensegrity structuresBody stands upright by compression due to gravity counteracted by tension from musclesSame for bridges and many other structures.

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Muscle

Tension

Mg

Bow and Arrow

Tension

Compression

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Muscle

Tension

Mg

TensegritySpectrinIn RBC100 nMNeurofilamentsCross-linkedIn frog axon

the CSK: smart designorienting along stress lines, filaments size themselves according to strength requirements: a conservative architectural practice. Thin supportingstruts connectingthick beams

Underneath the hoodLipid shell Actin networkCytosolFilamentsOrganellesNucleus

Lipid vesicles are ghost-likepipets suck up the vesiclesMiscibility allows intermingling

Plasma membraneLipid bilayer 30 ADielectric - capacitorAmphiphileSemi-permeableNo tensile but some shear strength

The cytoskeletonDecorated actin

Cartoon of membrane cross-section

CSK actin microfilamentsmicrotubulesintermediate filaments

ECM

Channels

Polymerized network:Spectrin in RBCs

Malines, BelgiumFibroblastTensegrity

Major FilamentsFilaments:Actin : 8 nMIntermediate 10nMMicrotubules:25 nM

Actin Intermediate Microtubules f ..filaments

3 types of filaments

Cellular Rods and Ropes

Filaments have different functionsSpectrin bends Microtubules are stiff

Cell Crawling

Visualizing actin-myosin motion

Types of motors

How does the CSK provide structure?

Some results are not compatible with tensegrity model Signals travel at speed of sound.

Structure by light & immunofluorescence

PMT

Fibroblasts are stained with Phallacidin green for F actin,Texas red for microtubules, and DAPI for nucleic acid.F actinmicrotubules

F actin is green with Phalloidin, G actin is red with Texasred. Nucleus has fewer stress fibers, but is thicker thanrest of cell, so red is diffuse. F actinG actin

Fibroblast dividing

Cells are Wiggly and Soft New ways to describe softness- difference between cookedand uncooked noodles.

thermal fluctuationsOf lipid vesicle:

Types of Loading

Swelling and Lysis to measure membrane strengthRBCsMuscle3%Frog

Pipet AspirationNeutrophils are WBCs involved in immune response.The source of cortical tension is unknown, but may befrom actin tangential to surface.

Unwinding of rubberseRubber ElasticityCollagen1 mm

Stress-strain varietiesseRubberJ Curveliquidunwinding

Solutes

Elasticity and safety at high strains

Mesangial cell area expansivityRubber-like

Common Quantities in Cytomechanics

Quantity

Units

Applications

Symbol

Stress, tension

Pascals, dynes/cm2

bars, rods, rubber, etc.

F

Pressure

same as above,

plus mm Hg, psi, millibars

Gases, liquids

P F

Membrane Tension

dynes/cm

thin shells, membranes

T = F/d

Where we are goingFeedback Regulation: Bioelectricity- eg. Heart, bone, cartilageOptics of cytoskeleton; immunofluoresc.Micromotors; Gels; piezo- & ferro-electricCell shape regulation, eg. Edema, tumorsTissue morphogenesis; osseointegrationEndothelial regulationWound healing

Common quantities

Strain

Extension

dimensionless

any deformable material

x-x

x

x/xo

Youngs Modulus

Pascals, dynes/cm2

E=

Viscosity

dynes-sec /cm2

Fluids, membranes

Shear stress

dynes /cm2

Fluids, membranes

ddt

The cytoskeleton is both internal and external

Fibroblast-myocyte interactionsFibroblastsMyocytes

Growth patterns vary in myocytes

Wall stress in a thick sphereTo find equilibrium forces:S Fup = SFdown

Membrane Tension

Cells will adhere to specific islands, properly coated.The traction force can be seen by the bending of the substrate. Microfabricated culture wells allow cell to make many E connections.

Shape Determined by Stress

Knock-out methods

Spectrin ActinMicrotubulesIntermediate FilamentsHeat CytochalasinNocodazoleAcyrlamide

Pulling Chromosomes out

CartilagepKa=pH +log[HA] [A-]Polymer charge determinesSwelling

Deformations of gel-cartilage

Swelling pressure = osmotic pressure- elastic (compressive) pressure

H20

Polymer-polymerIntra-polymerosmosis

FactorsCa++, pH

12

Tensegrity Industry

Designer foamZero Mean curvature

Percolation: theory of the CSK assembly Rule: network evolves by random connections between 2 active sites, each with some site occupation probability, p. A cluster is a set of occupied sites all of which are connected either horizontally or vertically, i.e. an occupied site belongs to a cluster if a member of the cluster is either above, below, left, or right of it. A spanning cluster has an element in both the top and bottom rows of the site matrix.

LA

NY

Hypothetical telephone network in the U.S.

ProteinsPrimary, secondary, tertiary, quaternary structuresMake filaments: rods, tubes,Flexural Stiffness

Polymer bendingassuming it is a thin rodLbendRqAt finite temperature, an otherwisestraight rod bends as it exchangesenergy with its environment. It bendsmore as T rises, like a noodle. Landau & Lifshitz,Theory of Elasticity, 1986. ?

What persistence length meansWhen L = ep , what happens at Earc= kT? Thus a rod of the persistence length is curved at 81 degrees when the thermal energy scale reaches kT.

If L > ep are highly convoluted and can assume many configurations.

Entropic springsLarge reeFew ConfigurationsSmall reeMany Config-urations

4-segment chain configurations24Applying a tension to the zero ree statereduces possible configurations to 10.S drops from ln(16) to ln (10). Hence tension translates to loss of entropy. tension

Effective spring constant for a convoluted chain near equilibrium:Have you seen a model of this?

TissueAggregate cells are more complicated. Many different types of connections, each with their own biochemical traffic patterns

Methods of cell regulation Signalling by mechanotransducers: current Molecular CSK regulators: Integrin Nuclear transcription: protein

ExerciseProve that a hollow design is advantageous for a microtubule. Assume an outer and inner radii of 14 and 11.5 nM, respectively, and compare this with a solid MT of the same outer radius. What is the most efficient way for proteins to gain rigidity, ie. on a per unit mass basis?

Review QuestionsThe cytoskeleton is made of (Select one)Filamentous proteinLipidActin, microtubules and microfilamentsExtracellular matrix (ECM)(a, b and c)(a and c)(all of the above)Integrin is a transmembrane peptide (True or False)

State a specific method or technique to "knock out" or remove a component of the cytoskeletonImmunofluorescence is a procedure to visualize specific molecules in a cell. The technique involves shining long wavelength light on the specimen, and seeing or detecting shorter wavelength light fluoresce. (True or false).