Cytoskeleton

• Form an elaborate interactive network of polymers of protein subunits held together by weak, noncovalent bonds

• Although they appear stationary in micrographs, they are highly dynamic structures capable of dramatic reorganization

• Recent study suggested that prokaryotes prokaryotes have proteins that carry cytoskeletal-like activities

Microtubules

• Long, hollow, and stiff unbranched tubes found in all eukaryotes that are distributed in the cytoplasm

• Functions in support, intracellular transport, and cell organization

• Composed of subunits of tubulin

Intermediate filaments

• Tough, ropelike fibers found in the nucleus and cytoplasm of animals

• Functions in structural support• Composed of a variety of proteins

Microfilaments

• Solid, thin structures found in the cytoplasm of eukaryotes that are often organized into a branching network

• Functions in motility and contractility• Composed of actin

Study of the cytoskeleton

Live-cell fluorescence imaging • allows researchers to directly observe

molecular processes in living cells—an approach known as live-cell imaging

FIGURE 9.4 Dynamic changes in length of microtubules withinan epithelial cell. The cell was injected with a small volume of

tubulin that had been covalently linked to the fluorescent dye rhodamine.After allowing time for the cell to incorporate the labeled

tubulin into microtubules, a small portion of the edge of the living cellwas examined under the fluorescence microscope.

In Vitro and In Vivo Single-molecule Assays• high-resolution video microscopy has led to

the development of in vitro motility assays• Single-molecule assays have allowed

researchers to make measurements that were not possible with standard biochemical techniques that average the results obtained from large numbers of molecules

In some of the earlier assays, microtubules were attached to a glass coverslip. Then, microscopic beads containing attached motor proteins were placed directly onto the microtubules using aimed laser beams. The laser beams are shone through the objective lens of a microscope, producing a weak attractive force near the point of focus. Because it can grasp microscopic objects, this apparatus is referred to as optical tweezers.

When ATP is present as an energy source, the movements of a bead along a microtubule can be followed by a video camera, revealing the size of individual steps taken by the motor protein. Focused laser beams can also be used to “trap” a single bead and determine the minute forces (measured as a few piconewtons, pN) generated by a single motor protein as it “tries” to move the bead against the force exerted by the optical trap

In this experiment a purified GFP-labeled kinesin molecule is seen to move processively along a microtubule whose plus end is labeled with a red fluorescent dye named Cy5.

The clarity of these images is made possible by the use of a specialized type of laser-based fluorescence microscopy called TIRF (total internal reflection microscopy)

Atomic force microscopy

-measures the mechanical properties of the cytoskeletal elements themselves. AFM is an instrument that uses a nanosized tip to probe the surface of a macromolecular specimen

- embed the tip of an AFM into a single intermediate filament and pull on the end or the middle of the filament to test its extensibility and tensile strength.

- a segment of filament can be mechanically stretched up to 3.5 times its normal length before it breaks into two pieces

Microtubules• components of a diverse array of structures, including

the mitotic spindle of dividing cells and the core of cilia and flagella

• outer diameter of 25 nm and a wall thickness of approximately 4 nm, and may extend across the length or breadth of a cell

• wall of a microtubule is composed of globular proteins arranged in longitudinal rows, protofilaments, that are aligned parallel to the long axis of the tubule

• 13 protofilaments aligned side by side in a circular pattern within the wall

• Each protofilament is assembled from dimeric building blocks consisting of one alpha-tubulin and one beta-tubulin subunit

• The tubulin dimers are organized in a linear array along the length of each protofilament

• All of the protofilaments of a microtubule have the same polarity. Consequently, the entire polymer has polarity. One end of a microtubule is known as the plus end and is terminated by a row of beta-tubulin subunits. The opposite end is the minus end and is terminated by a row of alpha- tubulin subunits(p325, d)

• contain additional proteins, called microtubule-associated proteins (MAPs)

• The binding of one of these MAPs to the surface of a microtubule connects microtubules to each other, thus maintaining their parallel alignment. MAPs generally increase the stability of microtubules and promote their assembly

Motor Proteins that Traverse the Microtubular skeleton

• convert chemical energy (stored in ATP) into mechanical energy, which is used to generate force or to move cellular cargo attached to the motor

• Collectively, motor proteins can be grouped into three broad superfamilies: kinesins, dyneins, and myosins. Kinesins and dyneins move along microtubules

Kinesins- discovered in 1985 by Ronald Vale and colleagues when they isolated a motor protein from the cytoplasm of squid giant axons- tetramer constructed from two identicalheavy chains and two identical light chains

• The routes followed by cytoplasmic vesicles and organelles are largely defined by microtubules, and members of the kinesin superfamily are strongly implicated as force-generating agents that drive the movement of this membrane bounded cargo

Cytoplasmic Dynein- discovered in 1963 as the protein responsible for the movement of cilia and flagella-a huge protein composed of two identical heavy chains and a variety of intermediate and light chains. Each dynein heavy chain consists of a large globular head(force generating agent) with an elongated projection (stalk)

• As a force-generating agent in positioning the spindle and moving chromosomes during mitosis

• As a minus end–directed microtubular motor with a role in positioning the centrosome and Golgi complex and moving organelles, vesicles, and particles through the cytoplasm

Intermediate filaments• strong, flexible ropelike fibers that provide

mechanical strength to cells that are subjected to physical stress, including neurons, muscle cells, and the epithelial cells that line the body’s cavities with a diameter of 10–12 nm

• chemically heterogeneous group of structures that, in humans, are encoded by approximately 70 different genes

• FIGURE 9.40 Cytoskeletal elements are connected to one another by protein cross-bridges. Electron micrograph of a replica of a small portion of the cytoskeleton of a fibroblast after selective removal of actin filaments. Individual components have been digitally colorized to assist visualization. Intermediate filaments (blue) are seen to be connected to microtubules (red) by long wispy cross-bridges consisting of the fibrous protein plectin (green).

Types of IFTypes I and II: Acidic Keratin and Basic Keratin, respectively. Produced

by different types of epithelial cells (bladder, skin, etc)Type III. Intermediate filaments are distributed in a number of cell

types, including: Vimentin in fibroblasts, endothelial cells and leukocytes; desminin muscle; glial fibrillary acidic factor in astrocytes and other types of glia, and peripherin in peripheral nerve fibers

Type IV Neurofilament H (heavy), M (medium) and L (low). Modifiers refer to the molecular weight of the NF proteins. Another type IV is "internexin" and some nonstandard IV's are found in lens fibers of the eye (filensin and phakinin).

Type V are the lamins which have a nuclear signal sequence so they can form a filamentous support inside the inner nuclear membrane. Lamins are vital to the re-formation of the nuclear envelope after cell division