- ex. polarized epithelial cells: certain organelles are arranged in defined order from apical to basal end of cell

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1 THE CYTOSKELETON - vertebrates: skeleton ; eukaryotic cells: cytoskeleton (analogous) - consists of hardened elements that support soft tissues of body - mediates bodily movements - 3 elements of cytoskeleton - protein subunits held together by weak, non covalent bonds - rapid assembly/disassembly dependent on complex cellular regulation - Microtubules - long, stiff, hollow unbranched tubes, inextensible tubes composed of subunits of protein tubulin. - for support, intracellular transport, cell organization - Microfilaments - solid, thinner structures, often organized into branching network composed of protein actin - for motility and contractility - Intermediate filaments - tough, flexible, extensible, rope-like fibers composed of variety of related proteins - for structural support 13.1 OVERVIEW OF MAJOR FUNCTIONS - dynamic scaffold providing structural support and determines shape of cell - internal framework responsible for positioning the various organelles inside the cell - ex. polarized epithelial cells: certain organelles are arranged in defined order from apical to basal end of cell - provide a network of tracks that direct movement of materials and organelles within the cell - ex. delivery of mrna to parts of cell, mebranous carriers from ER to Golgi complex, transport of neurotransmitter-containing vesicles in the nerve cell, peroxisome transport in mammalian cells - force-generating apparatus that moves the cell from one place to another - ex. movement via crawling or propelling through aqueous environment via microtubule-containing locomotor organelles: cilia & flagella - essential component of cell s division machinery (Spindle fibers) - cytoskeletal elements responsible for separating chromosomes in mitosis and splitting the parent cell in cytokinesis 13.2 STUDY OF CYTOSKELETON The Use of Live-Cell Fluorescence Imaging - fluorescence microscope: allowed researchers to directly observe molecular processes in living cells (live cell imaging) - fluorescently labeled proteins are synthesized in a cell as a fusion protein containing GFP. - Alternate approach: protein subunits are fluorescently labeled in vitro by covalent linkage to fluorescent dye - Fluorescence speckle microscopy: cytoskeletal elements are not uniformly labeled, instead contain irregularly spaced fluorescent speckles. These serve as fixed markers to follow dynamic changes in length/orientation of filament - Antibodies: useful in fluorescence microscopy because they can distinguish between isoforms of a protein The Use of In Vitro and In Vivo Single-Molecule Assays - In vitro motility assays: make possible to detect activity of protein molecule as molecular motor in real time - In vitro: kinesin motor was fused to GFP - Nanotechnology: development of nanomachines ( nm) able to perform activities in submicroscopic world - possible uses- medicine: machines introduced into body to carry specific tasks The Use of Fluorescence Imaging Techniques to Monitor the Dynamics of the Cytoskeleton - Fluorescence Recovery After Photobleaching (FRAP): Steps: - cell is injected w/ tubulin coupled to a fluorescent dye to express GFP-tubulin - fluorescence of the tubulin is bleached with laser beam. Specimen can then be followed - measure recovery signal into bleached zone 13.3 MICROTUBULE Structure & Composition - hollow, relatively rigid, tubular structures - found in nearly all eukaryotic cells - components for mitotic spindle of dividing cells & core of cilia and flagella - protofilaments: globular proteins arranged in longitudinal rows forming the microtubule wall - 13 aligned side by side in a circular manner, connected by non-covalent bonds - heterodimer- α-tubulin + β-tubulin = equal polarity per molecule but when together contributes to polarity of microtubule Microtubule-Associated Proteins (MAP s) - proteins found in microtubules prepared from living tissues - first MAPs to be identified -> classical MAPs - one domain/side attached to microtubule other projecting outward as a filament

2 - increase stability & promote assembly - Microtubule binding activity of MAPs controlled by add/remove of Phosphate group from amino acid residues - tau: MAP that is highly phosphorylated - implicated in the development of several fatal neurodegenerative disorders (Alzheimer's) caused by neurofibrillary tangles (tangling of unbound MAPs) - excessively phosphorylated = unable to bind to microtubule - FTDP-17: disease (inherited dementia), from mutation in the tau gene Structural Support and Organizers - stiff enough to resist force that might compress/bind the fiber - PROVIDES: mechanical support & structure - distribution of cytoplasmic microtubules helps determine shape of cell - Highly evident in cilia / flagella and axons of nerve cells - plant cells: influence shape through formation of cell wall - microfibrils are arranged perpendicular tp the long axis of the cell like hoops around a barrel. Because microfibrils prevent lateral expansions when turgor pressure is exerted by the fluid in the vacuoles it is directed to the end of th cell thus elongation. - Maintain internal organization of cells - treatment w/ microtubule-disrupting drugs can affect the location of membranous organelles (ER and Golgi) - treatment w/ nocodazole & colchicine (promote microtubule disassembly) can disperse golgi elements into diff regions - Drugs are removed = microtubules return Agents of Intracellular Motility - microtubules serve as tracks for motor proteins - Axonal Transport: Cell body in spinal cord; Axon terminal in fingertips - anterograde direction: structures and materials travelling from cell body->axon terminal (AWAY from cell body ANTEROGRADE) - retrogade direction: structures and materials travelling from axon terminal->cell body Motor Proteins - motor proteins convert chemical energy(stored in ATP) to mechanical energy - generates force to move cellular cargo - some types of cellular cargo- ribonucleic protein particles, vesicles, mitochondria, lysosomes, chromosomes, other cytoskeletal filaments - kinesins, dyneins move along microtubule, myosins move along microfilament, none in intermediate filament tracks(bec. non-polarized) - moves in unidirectional manner - mechanical cycle+chemical cycle = protein undergoes conformational change + utilizes energy to fuel activity - steps: ATP binds to motor -> ATP hydrolyzed -> Products (ADP + Pi) released -> ATP binds to motor - binding & hydrolyzing -> drives a power stroke that moves the motor a precise number of nanometers - Kinesins - Found in all eukaryotic cells - Made of a tetramer (two identical heavy chains and two identical light chains) - Has a pair of globular heads (motor domains) that bind a microtubule (ATP-hydrolyzing force-generating engines) - moves toward (+) end - plus-end directed microtubular motor - in axons: (-) end facing cell body, thus kinesins moves in the anterograde direction - Kinesins movement is as a velocity proportional to ATP concentration, thus low ATP = slow. - movement is hand over hand like climbing a rope. Each step (8nm) powered by 1 ATP - The two heads behave in a coordinated manner -> when one binds causing a conformational change, the other moves forward to the next binding site on the protofilament - kinesins-1: conventional kinesins - discovered kinesins-14: move in the opposite direction. Due to difference in adjacent neck region of the two proteins - kinesins-13: does not move at all but instead binds to either side of the microtubule and causes depolarization. Thus is thin kinesins is also called depolymerase - Kinesins superfamilies tend to move vesicles and organelles in an outward direction towards the cell s plasma membrane - Dynein - Responsible for cilia and flagella movement - Cytoplasmic dyneins are present throughout the animal kingdom - sketchy kung meron ba sa plants.

3 - Whereas each of us have several kinesins and myosins we can only survive with 2 dyneins - one of which is responsible for most transportation operations - Composed of two identical heavy chains and a variety of intermediate light chains - Each heavy chain consists of a large globular head (force-generating engine) with an elongated projection / stalk with a microtubular binding site at its tip. - moves towards the (-) end, opposite that of kinase. - roles: - force generating agent in position spindle fiber and moving chromosomes (mitosis) - (-)end-directed microtubular motor with a role in position the centromeres and Golgi complex and moving organelles, vesicles, and particles through the cytoplasm - Known for RETROGRADE MOVEMENT of membranous organelles, and ANTEROGRADE MOVEMENT of microtubules. - do not directly interact with cargo -> require an intervening multisubunit adaptor called dynactin - also regulates dynein activity - helps bind motor protein to microtubule w/ increased processivity - kinesins and dyneins move similar materials in opposite directions over the same railway network Microtubule-Organizing Centers (MTOCs) - Assembly of microtubules from α-tubulin and β-tubulin - PHASE 1: Nucleation - small portion of microtubular is formed - PHASE 2: Elongation - rapid phase that occurs in association with a variety of specialized structures (MTOCs) - Centrosomes - best studied MTOCs are found here. - composed of two barrel-shaped centrioles - these centrioles contains 9 evenly shape fibers which appear in cross sections as a band of three microtubules (A,B, and C) wherein only the A tubule is a complete microtubule - arranged in a manner that gives centriole pinwheel like appearance - Function - Control the number of microtubules - Control polarity - Regulate the number of protofilaments - Regulate time of assembly - All MTOCs contain (gamma)ɣ-tubulin which is a critical component in microtubule nucleation - Microtubules of the mitotic spindle are sensitive to disassembly - Induced by colchicine, vinblastine, - Taxol(catharanthus roseus):drug - stops the dynamic activity of microtubules - Prevents cell from assembling new microtubular structures (used in cancer therapy) - once disrupted cells will not proceed to division - Microtubules stable in cilia and flagella - Dissect microtubule you can find the axoneme: core of the cilium, contains an array of microtubules that run longitudinally through the entire organelle The Human Perspective: Role of Cilia in Development of Disease TO FOLLOW IN LIFE INTERMEDIATE FILAMENTS (IFs) - only found in animal cells - strong, flexible, rope-like fibers - provide mechanical strength to cells under physical stress - chemically heterogeneous group of structures (encoded by 70 genes in humans) - plectin: thin, wispy cross-bridges connecting IF to other cytoskeletal filaments. Types and Functions - each kind of IFs have tissue-specific functions, which mean some IFs are more important in some cells than other - keratin filaments: constitute the primary structural proteins of epithelial cells, epidermal cells, liver hepatocytes, pancreatic acinar cells - neurofilaments: intermediate filaments oriented parallel to the nerve cell axon - composed of 3 proteins (full word of the protein acronym not mentioned in book)

4 - NF-L - NF-H -have sidearms that project outward from neurofilament - NF-M -have sidearms that project outward from neurofilament - sidearms- maintain proper spacing between parallel neurofilaments of the axon - aggregates of of NFs = neurodegenerative diseases (Amyotrophic lateral sclerosis [ALS], Parkinson s Disease) - desmin: maintains the alignment of the myofibrils of a muscle cell, absence of these makes cells really fragile 13.5 MICROFILAMENTS - composed of globular subunits of actin - if w/ ATP, actin monomers polymerize to form helical filament - Actin: 2 stranded w/ 2 helical grooves along the whole length - synonyms: Actin filament, microfilament, F-actin - entire microfilament has polarity - the ends, F-actin have different structures and dynamic properties - can be organized into highly ordered arrays, loos ill-defined networks, or tightly anchored bundles - Myosin: a motor protein, that moves toward the (+) end of actin filament - all myosin have same characteristic motor (head) domain - contains a site that binds to actin filament & a site that binds/hydrolyzes ATP - have variety of low-molecular-weight chains (light chains) - Conventional Myosins(Type II): primary motors for muscle contraction, splitting cell in two (cell division), generating tension at focal adhesions, cell migration, has two heads - Unconventional Myosin(Type I): only has a single head, precise role in cellular activity is unclear - Unconventional Myosin(Type V): dimeric, two heads, long = takes very large steps - Unconventional Myosin(Type VI): thought to be involved in formation of clathrin-coated vesicles at plasma membrane, movement of uncoated vesicles to early endosomes, anchoring of membranes to actin filaments 13.6 MUSCLE CONTRACTILITY Skeletal Muscles: Under voluntary control - more appropriately called muscle fibers - Longitudinal cross section reveals presence of microfibrils, cables made up of hundreds of thinner, cylindrical strands. - Each microfibril consists of a repeating linear array of contractile units called, sarcomeres - Each sarcomere in turn exhibits banding patterns (striated appearance) -> banding patterns are a result of partial overlap between 2 distinct types of filament thin filament and thick filament. - a sarcomere spans from one Z-line to another Z-line which contains several dark & light bands - I band : lightly stained and located on the outer edges (both sides). Consists of only thin filament - A band: densely stained and located between the outer I band - H zone: the center area, lightly stained. Consists of only thick filaments - M line: a line down the center of the H band - note: the region of overlap of thin and thick filaments is at the part of the A band on either side of the H zone. Sliding Filament Model of Muscle Contraction - All skeletal muscles operate by shortening, no other means to perform work - Units of shortening are the sarcomeres - PROCESS: As a muscle fiber shortens, the A band remains at constant length. While H zone and I band decrease in width until they disappear altogether. Simultaneous to this is the inward movements of the Z line until they contract the outer edges of the A zone (Figure page 541) - It was proposed that the shortening of individual sarcomeres did not result from the shortening of the filaments, but rather their sliding over one another. Composition and Organization of Thick and Thin Filaments - Thin Filament Components - Actin - Tropomyosin: elongated molecule, fits securely into groove within thin filaments - Troponin: composed of three subunits, each having an important distinct role. Spaced along the thin filaments in such a way that it makes contact with both tropomyosin and actin components in the filament - Thick Filament Components - composed of several hundred myosin II molecules together with small amounts of other proteins - the polarity of thick filaments is reversed at the center of the sarcomere

5 - Titin : largest protein yet to be discovered in any organism originate from M line and extend until Z line. - function: prevent sarcomere from being pulled apart during muscle stretching - maintain myosin filaments in their proper position. Molecular Basis of Contraction - Myosin heads: force-generating component of the muscle fibers - Contraction - myosin head extend outward and binds to the actin filaments (1 head : 4 filaments) thus forming a cross bridge - Muscle myosins is a nonprocessive arm causing the attached actin filaments to slide a much greater distance than would otherwise be possible.