The Cytoskeleton Assembly and Dynamic Structure of Cytoskeletal Filaments Pages 965-991

The Cytoskeleton Green = Microtubules Red = Actin Blue = Coomassie Blue stain

Functions of the Cytoskeleton -Pull chromosomes apart at mitosis and then splits the dividing cell into two -intracellular trafficing of organelles and other proteins -support of the plasma membrane -allows the cell to bear stresses and strains -allows cells to swim (sperm) or crawl (fibroblasts) -provides machinery for muscle contraction -allows neurons to extend axons and dendrites

Types of Cytoskeletal Filaments 1. Actin Filaments -determine the shape of the cell’s surface -necessary for whole-cell locomotion -diameter: 5-9 nm 2. Intermediate Filaments -provide mechanical strength and resistance shear stress -diameter: 10 nm 3. Microtubules -determine the positions of membrane-enclosed organelles -directs intracellular transport -diameter: 25 nm

Changes in the Cytoskeleton Microtubules Actin Red = Actin Green = Microtubules

Actin and Microtubules in Drosophila Embryos Actin – Red Microtubules – Green Division every 10 min

Neutrophil Movement Assembly and disassembly of the actin filaments allows the cell to change directions

Similarities between the Types of Filaments 1. They form as helical assemblies of subunits 2. They all self-associate using a combination of end- to-end and side-to-side protein contacts 3. Assembly and disassembly can occur rapidly 4. Accessory proteins regulate the spatial distribution and dynamic behavior of the filaments

Cytoskeletal Structure -Assymetric -Characteristic Shape

Polarity of Actin in Yeast

Rapid Reorganization of the Cytoskeleton

Protofilament Formation Protofilament – long linear strings of subunits joined end-to-end Composed of globular subunits that make a similar number of longitudinal and lateral bonds

Actin and Microtubule Formation

Intermediate Filaments Composed of fibrous subunits that make more lateral bonds than longitudinal bonds

3 Stages of Polymerization Nucleation – Subunits must assemble into an initial aggregate that is stabilized by many subunit-subunit interactions -This is the rate-limiting step in polymerization -Special proteins catalyze nucleation at specific sites

Microtubule Structure GTP/GDP Tightly bound 13 protofilaments

Actin Filament Structure 2 protofilaments

Preferential Growth of Microtubules -Polarity of Microtubules and Actin Filaments is created by the parallel orientation of its subunits -Alpha units are exposed at + end and beta subunits at the – end -The + end is more dynamic, growing and shrinking faster, while the – end is slower

Treadmilling in a Living Cell Microtubules

Treadmilling of Actin T form contains ATP D form contains ADP Treadmilling – Subunits are added at the + end while being removed at the - end

Dynamic Instability of Microtubules Cc(D) > Cc(T) -The D form leans more readily toward disassembly -The T form leans more readily towards assembly Dynamic Instability – the rapid interconversion between a growing and shrinking state at a uniform free subunit concentration

GTP Hydrolysis Causes Structural Changes GTP- straight filaments GDP – curved filaments

Structural Changes in Microtubules Curved filaments (GDP) doesn’t allow for as many interactions between protofilaments

Dynamic Instability in a Living Cell Treadmilling and dynamic instability large amounts of energy are used, but it gives the cell spatial and temporal flexibility in response to its environment

FtsZ, a Tubulin Homologue in Procaryotes -Essential in cell division, a band of FtsZ protein forms at the site of separation, where the new cell wall is to form -Constriction and disassembly of the FtsZ band through GTP hydrolysis helps to pinch the two daughter cells apart

Actin Interactions -Actin is found in all eucaryotic cells -humans have 6 different actin genes, alpha actin is expressed in only muscle cells while beta and gamma are in almost all nonmuscle cells -Tubulin is also found in all eucaryotic cells and there are also multiple forms of the subunits

Intermediate Filament Subunits -no overall polarity

Intermediate Filament Construction -They are easily bent but extremely difficult to break -Protein phosphorylation probably regulates their disassembly

Mechanical Properties of Cytoskeletal Filaments

Keratin Filaments in Epithelial Cells -About 20 different types found in human epithelial cells and about 10 more that are specific to hair and nails -Keratin filaments are made of equal numbers of type I (acidic) and type II (neutral/basic) chains -Usually they are crosslinked by disulfide bonds

Blistering of the Skin due to Mutated Keratin Epidermolysis bullosa simplex – the skin blisters in response to even slight mechanical stress, which ruptures the basal cells