Cytoskeleton DR PALLEE SHREE 1. DETERMINE THE SHAPE OF THE CELLS AND PROVIDE STRENGTH Cell shape & strength • Actin filaments are highly concentrated at the periphery of the cell where they form a 3D network beneath the plasma membrane • This network of actin filaments and associated actin-binding proteins and form cell cortex which determines cell shape and also help in cell surface activities Cont…. • The cortical actin cytoskeleton is responsible for distinctive shape as biconcave discs • As erythrocytes lack microtubules and intermediate filaments • The principal advantage of red blood cells for these studies is that they don't contain internal organelles, so their plasma membrane and associated proteins can be easily isolated Actin-binding protein of erythrocytes- spectrin • The beta chain has a single actin- • Actin-binding protein- spectrin binding domain at its amino associate with short actin terminus. filaments • Link between the spectrin-actin network and the plasma • Result in the spectrin-actin membrane is provided by a network that forms the cortical protein called ankyrin cytoskeleton of RBC which binds both to spectrin and to a transmembrane protein • Spectrin is a member of the large called band 3. calponin family of actin-binding • An additional link between the spectrin-actin network and the • Spectrin is a tetramer consisting plasma membrane is providedby of two distinct polypeptide chains protein 4.1 called a and beta Structure of spectrin (just for understanding) Association of the erythrocyte cortical cytoskeleton with the plasma membrane Cont… • Member of the calponin family, filamin constitutes a major link between actin filaments and the plasma membrane of blood platelets. • Additional member of the calponin family, dystrophin in muscles • Absent or abnormal in patients cause muscular dystrophy 2. HELP IN ESTABLISHING CONTACTS WITH ADJACENT CELLS OR EXTRACELLULAR MATRIX Most cells have specialized regions of the plasma membrane that form contacts with adjacent cells, the extracellular matrix or with other substrata such as the surface of a culture dish. a. Establishing contacts with extracellular matrix • Regions of attachment sites is contributed by for bundles of actin filaments that anchor the cytoskeleton of cell to areas of cell contact. • The best Example: fibroblasts maintained in tissue culture. • The fibroblasts attach to this extracellular matrix on the culture dish via the binding of transmembrane proteins called integrins. • The sites of attachment are discrete regions called focal adhesions that also serve as attachment sites for large bundles of actin filaments called stress fibers. (Refer diagram in the next slide) Attachment of stress fibers to the plasma membrane at focal adhesions b. Actin cytoskeleton is anchored to regions of cell-cell contact (adherens junctions) • In sheets of epithelial cells, these junctions form a continuous belt-like structure called an adhesion belt around each cell • Contact between cells at adherens junctions is mediated by transmembrane proteins called cadherins. • The cadherins form a complex with cytoplasmic proteins called catenins, which associate with actin filaments. (Refer diagram) Attachment of actin at adherence junctions 3. ACTIN HELP IN PROTRUSIONS OF THE CELL SURFACE Most of these cell surface extensions are based on actin filaments, which are organized into either relatively permanent or rapidly rearranging bundles or networks. a. Permanent Protrusions of the Cell Surface • Best-characterized of these actin-based cell surface protrusions are microvilli on epithelial cells lining intestine they form brush border • Abundant on the surfaces of cells involved in absorption • Another example of specilized microvilli is stereocilia of auditory hair cells, are responsible for hearing by detecting sound vibrations. • Microvilli -parallel bundles of 20 to 30 actin filaments in these bundles are cross-linked in part by fimbrin and villin • Along their length, the actin bundles of microvilli are attached to the plasma membrane by lateral arms consisting of the calcium-binding protein calmodulin in association with myosin l b.Transient surface protrusions • Pseudopodia are extensions of moderate width, based on actin filaments cross-linked into a three- dimensional network • Lamellipodia are broad, sheetlike extensions at the leading edge of fibroblasts, which similarly contain a network of actin filaments • Many cells also extend microspikes or filopodia 4. ACTIN HELP IN MUSCLE CONTRACTION Muscle Contraction • Skeletal muscles are bundles of muscle fibers • Most of the cytoplasm consists of myofibrils, which are cylindrical bundles of two types of filaments: thick filaments of myosin (about 15 run in diameter) and thin filaments of actin (about 7 nm in diameter). • Each myofibril is organized as a chain of contractile units called sarcomeres, which are responsible for the striated appearance of skeletal and cardiac muscle. Structure of muscle cells Sarcomere • The ends of each sarcomere are defined by the Z disc. • Within each sarcomere, dark bands (called A bands because they are anisotropic when viewed with polarized light) alternate with light bands (called I bands for isotropic). • The I bands contain only thin (actin) filaments, whereas the A bands contain thick (myosin) filaments. • The myosin and actin filaments overlap in peripheral regions of the A band, whereas a middle region (called the H zone) contains only myosin. Muscle contraction • The basis for understanding muscle contraction is the sliding filament model, first proposed in 1954 both by Andrew Huxley and Ralph Niedergerke and by Hugh Huxley and Jean Hanson • During muscle contraction each sarcomere shortens, bringing the Z discs closer together. • There is no change in the width of the A band, but both the I bands and the H zone almost completely disappear. • These changes are explained by the actin and myosin filaments sliding past one another so that the actin filaments move into the A band and H zone. • Muscle contraction thus results from an interaction between the actin and myosin filaments that generates their movement relative to one another. • The molecular basis for this interaction is the binding of myosin to actin filaments, allowing myosin (motor protein convert chemical energy to mechanical) to function as a motor that drives filament sliding. Sliding filament model (sarcomere) Association of tropomyosin and troponins with actin filaments Ca2+-binding protein calmodulin ***Refer to pdf shared for more detail. 5. NON MUSCULAR MYOSIN AND ACTIN LEADS TO CYTOKINESIS 5. Non muscular myosin and actin leads to cytokinesis Contractile assemblies in nonmuscle cells • The most dramatic example of actin-myosin contraction in nonmuscle cells, is provided by cytokinesis • Toward the end of mitosis in yeast and animal cells, a contractile ring consisting of actin filaments and myosin assembles just underneath the plasma membrane. • Its contraction pulls the plasma membrane progressively inward, constricting the center of the cell and pinching it in two. Interestingly, the thickness of the contractile ring remains constant as it contracts, implying that actin filaments disassemble as contraction proceeds. • The ring then disperses completely following cell division • In nonmuscle cells and in smooth muscle, however, contraction is regulated primarily by phosphorylation of one of the myosin light chains called the regulatory light chain 6.FORMATION OF PROTRUSIONS AND CELL MOVEMENT 6.Formation of Protrusions and Cell Movement • The movement of cells across a surface represents a basic form of cell locomotion employed by a wide variety of different kinds of cells. Examples includes: The crawling of amoebas The migration of embryonic cells during development The invasion of tissues by white blood cells to fight infection The migration of cells involved in wound healing The spread of cancer • All of these movements are based on local specializations and extensions of the plasma membrane driven by the dynamic properties of the actin cytoskeleton. Cell movement or extension involves a coordinated cycle of movements • First, cells must develop an initial polarity via specialization of the plasma membrane or the cell cortex. • • Second, protrusions such as pseudopodia, lamellipodia, or filopodia must be extended to establish a leading edge of the cell. These extensions must then attach to the substrahtm across which the cell is moving. • Finally, during cell migration the trailing edge of the cell must dissociate from the substratum and retract into the cell body. Intermediate Filaments • Intermed ate filaments have diameters between 8 and 11 nm • Not involved in cell movements instead, play a structural role by providing mechanical strength to cells • Intermediate filaments are apolar Structure • Intermediate filaments are composed of a variety of proteins that are expressed in different types of cells • More than 65 different intermediate filament proteins have been identified • These proteins are classified into six groups based on similarities between their amino acid sequences (refer table) Classes of intermediate filament and their functions Functions Structure of Intermediate Filaments Assembly of intermediate filament Step 1 Formation of dimers in which the central rod domains of two polypeptide chains are wound around each other in a coiled-coil structure Step 2 The dimers of cytoskeletal intermediate filaments then associate in a staggered antiparallel fashion to form tetramers