Cytoskeletal Systems

Chapter 15

Lectures by Kathleen Fitzpatrick Simon Fraser University

• Microtubules are the largest of the cytoskeletal components of a cell

• There are two types of microtubules

• They are involved in a variety of functions in the cell

- Maintaining axons - Formation of mitotic and meiotic spindles - Maintaining or altering cell shape - Placement and movement of vesicles

• Axonemal microtubules include the organized and stable microtubules found in structures such as

- Cilia - Flagella - Basal bodies to which cilia and flagella attach

• The axoneme, the central shaft of a cilium or flagellum, is a highly ordered bundle of MTs

• MTs are straight, hollow cylinders of varied length that consist of (usually 13) longitudinal arrays of polymers called protofilaments

• The basic subunit of a protofilament is a heterodimer of tubulin, one a-tubulin and one b- tubulin

• These bind noncovalently to form an ab- heterodimer, which does not normally dissociate

• a and b subunits have very similar 3-D structure, but only 40% amino acid identity

• Each has an N-terminal GTP binding domain, a central domain to which colchicine can bind, and a C-terminal domain that interacts with MAPs (microtubule-associated proteins)

• All the dimers in the MT are oriented the same way

• Because of dimer orientation, protofilaments have an inherent polarity

• The two ends differ both chemically and structurally

• Most organisms have several closely related genes for slight variants of a- and b-tubulin, referred to as isoforms

• Cytoplasmic MTs are simple tubes, or singlet MTs, with 13 protofilaments

• Some axonemal MTs form doublet or triplet MTs

• Doublets and triplets contain one 13- protofilament tubule (the A tubule) and one or two additional incomplete rings (B and C tubules) of 10 or 11 protofilaments

• MTs form by the reversible polymerization of tubulin dimers in the presence of GTP and Mg2+

• Dimers aggregate into oligomers, which serve as “nuclei” from which new MTs grow

• This process is called nucleation; the addition of more subunits at either end is called elongation

• MT formation is slow at first, the lag phase, due to the slow process of nucleation

• The elongation phase is much faster

• When the mass of MTs reaches a point where the amount of free tubulin is diminished, the assembly is balanced by disassembly; the plateau phase

• Microtubule assembly in vitro depends on concentration of tubulin dimers

• The tubulin concentration at which MT assembly is exactly balanced by disassembly is called the critical concentration

• MTs grow when the tubulin concentration exceeds the critical concentration and vice versa

© 2012 Pearson Education, Inc. Addition of Tubulin Dimers Occurs More Quickly at the Plus Ends of Microtubules • The two ends of an MT differ chemically, and one can grow or shrink much faster than the other • This can be visualized by mixing basal bodies (structures found at the base of cilia) with tubulin heterodimers • The rapidly growing MT end is the plus end and the other is the minus end

• The plus and minus ends of microtubules have different critical concentrations

• If the [tubulin subunits] is above the critical concentration for the plus end but below that of the minus end, treadmilling will occur

• Treadmilling: addition of subunits at the plus end, and removal from the minus end

• Colchicine binds to tubulin monomers, inhibiting their assembly into MTs and promoting MT disassembly

• Vinblastin, vincristine are related compounds

• Nocodazole inhibits MT assembly, and its effects are more easily reversed than those of colchicine

• These drugs are called antimitotic drugs because they interfere with spindle assembly and thus inhibit cell division

• They are useful for cancer treatment (vinblastine, vincristine) because cancer cells are rapidly dividing and susceptible to drugs that inhibit mitosis

• Taxol binds tightly to microtubules and stabilizes them, causing a depletion of free tubulin subunits

• It causes dividing cells to arrest during mitosis

• It is also used in cancer treatment, especially for breast cancer

© 2012 Pearson Education, Inc. Microtubules Originate from Microtubule- Organizing Centers Within the Cell • MTs originate from a microtubule-organizing center (MTOC) • Many cells have an MTOC called a centrosome near the nucleus • In animal cells the centrosome is associated with two centrioles, surrounded by pericentriolar material

• Centriole walls are formed by 9 pairs of triplet microtubules

• They are oriented at right angles to each other

• They are involved in basal body formation for cilia and flagella

• Cells without centrioles have poorly organized mitotic spindles

• Centrosomes have large ring-shaped protein complexes in them; these contain g-tubulin (along with gamma tubulin ring proteins: GRiPs)

• g-tubulin ring complexes (g-TuRCs) nucleate the assembly of new MTs away from the centrosome

• Loss of g-TuRCs prevents a cell from nucleating MTs

• MTOCs nucleate and anchor MTs

• MTs grow outward from the MTOC with a fixed polarity—the minus ends are anchored in the MTOC

• Because of this, dynamic growth and shrinkage of MTs occurs at the plus ends, near the cell periphery

• Cells regulate MTs with great precision

• Some MT-binding proteins use ATP to drive vesicle or organelle transport or to generate sliding forces between MTs

• Others regulate MT structure

• Microfilaments are the smallest of the cytoskeletal filaments

• They are best known for their role in muscle contraction

• They play a role in cell migration, amoeboid movement, and cytoplasmic streaming

• Development and maintenance of cell shape (via microfilaments just beneath the plasma membrane at the cell cortex)

• Structural core of microvilli

• Actin is a very abundant protein in all eukaryotic cells

• Once synthesized, it folds into a globular-shaped molecule that can bind ATP or ADP (G-actin; globular actin)

• G-actin molecules polymerize to form microfilaments, F-actin

• Actin is highly conserved, but there are some variants

• Actins can be broadly divided into muscle-specific actins (a-actins) and nonmuscle actins (b- and g-actins)

• b- and g-actin localize to different regions of a cell

• G-actin monomers can polymerize reversibly into filaments with a lag phase, and elongation phase, similar to tubulin assembly

• F-actin filaments are composed of two linear strands of polymerized G-actin, wound into a helix

• All the actin monomers in the filament have the same orientation

• Myosin subfragment 1 (S1) can be incubated with microfilaments (MFs)

• S1 fragments bind and decorate the actin MFs in a distinctive arrowhead pattern

• The plus end of an MF is called the barbed end and the minus end is called the pointed end, because of this pattern

• The polarity of MFs is reflected in more rapid addition or loss of G-actin at the plus end than the minus end

• After the G-actin monomers assemble onto a microfilament, the ATP bound to them is slowly hydrolysed

• So, the growing MF ends have ATP-actin, whereas most of the MF is composed of ADP- actin

• Cytochalasins are fungal metabolites that prevent the addition of new monomers to existing MFs

• Latrunculin A is a toxin that sequesters actin monomers and prevents their addition to MFs

• Phalloidin stabilizes MFs and prevents their depolymerization

© 2012 Pearson Education, Inc. Actin-Binding Proteins Regulate the Polymerization, Length, and Organization of Actin • Cells can precisely control where actin assembles and the structure of the resulting network

• They use a variety of actin-binding proteins to do so

• Control occurs at the nucleation, elongation, and severing of MFs, and the association of MFs into networks

• Intermediate filaments are the most stable and least soluble cytoskeletal components and are not polarized

• An abundant intermediate filament (IF) is keratin, an important component of structures that grow from skin in animals

• IFs may support the entire cytoskeleton

• IFs differ greatly in amino acid composition from tissue to tissue

• They are grouped into six classes

• Class I: acidic keratins

• Class II: basic or neutral keratins

• Proteins of classes I and II make up the tonofilaments found in epithelial surfaces covering the body and lining its cavities

• Class III: includes vimentin (connective tissue), desmin (muscle cells), and glial fibrillary acidic (GFA) protein (glial cells)

• Class IV: These are the neurofilament (NF) proteins found in neurofilaments of nerve cells

• Class V: includes the nuclear lamins A, B, and C that form a network along the inner surface of the nuclear membrane

• Class VI: Neurofilaments in the nerve cells of embryos are made of nestin

• Animal cells can be distinguished based on the types of IF proteins they contain—intermediate filament typing

• IF proteins are fibrous rather than globular

• All have a homologous central rodlike domain conserved in size, secondary structure, and to some extent, in sequence

• Flanking the central helical domain are N- and C-terminal domains that differ greatly among IF proteins

• Cellular architecture depends on the unique properties of the cytoskeletal elements working together

• MTs resist bending when a cell is compressed whereas MFs serve as contractile elements that generate tension

• IFs are elastic and can withstand tensile forces

• IFs are important structural determinants in many cells and tissues; they are thought to have a tension-bearing role

• IFs are not static structures; they are dynamically transported and remodeled

• The nuclear lamina, on the inner surface of the nuclear envelope, disassemble at the onset of mitosis and reassemble afterward

• Plakins are linker proteins that connect intermediate filaments, microfilaments, and microtubules

• One plakin, called plectin, is found at sites where intermediate filaments connect to MFs and MTs