Week 6 Lecture Notes
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Date Created: 10/08/16
Week 6 Class Notes 9/29-10/6 Big Picture: We are now going to be talking about the cytoskeleton and we will break down the big three components of microtubules and look at them with great detail. The Cytoskeletal System as a Whole The cytoskeleton is a network of interconnected filaments and tubules extending through the cytosol It plays roles in cell movement and division It is dynamic and changeable The Big 3: Microfilament, Microtubules, Intermediate Filaments (all of these guys are polymers Microtubules (Highways of the cell) (Largest) Functions Organization of intracellular organelles and transport of vesicles (motor proteins) Beating of cilia and flagella Nerve cell, red blood cell, and flagellar structure Alignment and separation of chromosomes during mitosis and meiosis Characteristics Unstable Form o mitotic apparatus o short lived o assembles quickly and then dissembles just as quick Stable Form o long lived o remains polymerized for a long time o An example is cilia on egg: moves it along the oviduct; if you don’t have this you will be sterile 2 Types of MT o Cytoplasmic (Unstable) Functions: Maintaining axons Formation of mitotic and meiotic spindles Maintaining or altering cell shape Placement and movement of vesicles o Axonemal microtubules (Stable) Functions in: 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 Structure (How they are made) 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 α-tubulin and one β-tubulin These bind noncovalently to form an αβ-heterodimer, which does not normally dissociate IMPORTANT: IF YOU ARE GOING α-α OR β-β THEN YOU CAN CHANGE THE GTP. BUT IF YOU GO α-β, THEN YOU CAN’T CAHNGE GTP!!!!!! Subunit Structure o Each one is 3-D I identical by 40% matched AA wise o 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) o All the dimers in the MT are oriented the same way o Because of dimer orientation, protofilaments have an inherent polarity o The two ends (called the plus end and the minus end) differ both chemically and structurally o Most organisms have several closely related genes for slight variants of α- and β-tubulin, referred to as tubulin isoforms Don’t have to know isoforms How they form o MTs form by the reversible polymerization of tubulin dimers in the 2+ presence of GTP and Mg o Dimers aggregate into oligomers, which serve as “seeds” from which new MTs grow o This process is called nucleation; the addition of more subunits at either end is called elongation o THIS IS IMPORTANT THE AMOUNT OF DIMERS NEEDED FOR AN MT TO GROW NEEDS A THREASHOLD VALUE!!!!!!!!! MT FORMATION REQUIRES A SPECIFIC NUMBER OF MOLECULES TO START THE PLUS END THRESHOLD VALUE IS LOWER THAN THE MINUS END. THUS THE MT WILL GROW IN THIS DIRECTION!!!!!! THIS IS BECAUSE THERE IS A GREATER GTP BINDING CAPACITY!!! Facts you need to know about formation o Free tubulin exists as a/b dimer o b(GTP) dimer will add onto (+) end o Addition of next dimer will trigger GTP hydrolysis in b-subunit o Spontaneous nucleation is rare in vivo; need MTOCs When the mass of MTs reaches a point where the amount of free tubulin is diminished, the assembly is balanced by disassembly; this is known as the plateau phase The elongation phase is much faster MT formation is slow at first because the process of nucleation is slow; this period is known as the lag phase Some Definitions you need to know Centrosome- is two centrioles Critical Concentration- The tubulin concentration at which MT assembly is exactly balanced by disassembly o MTs grow when the tubulin concentration exceeds the critical concentration and vice versa Treadmilling addition of subunits at the plus end, and removal from the minus end If the free tubulin concentration is above the critical concentration for the plus end but below that of the minus end, treadmilling will occur Microtubules Can Form as Singlets, Doublets, or Triplets Cytoplasmic MTs are simple tubes, or singlet MTs, with 13 protofilaments (this is like what we have been talking about Some axonemal MTs form doublet or triplet MTs (Cilia, Flagella) 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 (basal bodies, centrioles) Drugs can affect the formation of MT Colchicine binds to tubulin monomers, inhibiting their assembly into MTs and promoting MT disassembly (cancer treatment) o from Meadow Saffron o sequesters tubulin dimers o treatment for gout, depolymerizes white blood cells, cannot get to site of gout, reduces inflammation Vinblastine, vincristine are related compounds Nocodazole inhibits MT assembly, and its effects are more easily reversed than those of colchicine (Cancer treatment) Antimitotic Drugs o These drugs are called antimitotic drugs because they interfere with spindle assembly and thus inhibit cell division o They are useful for cancer treatment (vinblastine, vincristine) because cancer cells are rapidly dividing and susceptible to drugs that inhibit mitosis o Taxol binds tightly to microtubules and stabilizes them, causing a depletion of free tubulin subunits from Pacific Yew Tree stabilizes microtubules treatment for cancers, prevents mitosis o It causes dividing cells to arrest during mitosis o It is also used in cancer treatment, especially for breast cancer Instability of MT GTP and its relation to this whole process o Each tubulin heterodimer binds two GTP molecules; α-tubulin binds one, and β-tubulin binds a second o The GTP bound to the β-subunit is hydrolyzed to GDP after the heterodimer is added to the MT o GTP is needed to promote heterodimer interactions and addition to MTs, but its hydrolysis is not required for MT assembly Dynamic Instability Model (Catastrophe) o one population of MTs grows by polymerization at the plus ends, whereas another population shrinks by depolymerization o Growing MTs have GTP at the plus ends, and shrinking MTs have GDP o The GTP cap at the plus end prevents subunit removal o If GTP-tubulin is high, it is added to an MT quickly, creating a large GTP- tubulin cap o If the concentration falls, the rate of tubulin addition decreases o At a sufficiently low GTP-tubulin, the rate of GTP hydrolysis exceeds the rate of subunit addition, and the cap shrinks o When it pulls apart, it pulls apart like a banana. Proteofiliment by proteofiliment. Catastrophe and Rescue o If the GTP cap disappears altogether, the MT becomes unstable, and loss of GDP-bound subunits is favored o If there are no dimers then catastrophe will occur because any dimmer present cannot be added by the time the β- GTP is hydrolyzed. o Individual MTs can go through periods of growth and shrinkage; a switch from growth to shrinkage is called microtubule catastrophe o A sudden switch back to growth phase is called microtubule rescue Centriole Structure Centriole walls are formed by nine 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 γ- TubulinRC (ring complex) Centrosomes have large ring-shaped protein complexes in them; these contain γ-tubulin γ-tubulin is found only in centrosomes γ-tubulin ring complexes (γ-TuRCs) nucleate the assembly of new MTs away from the centrosome Loss of γ-TuRCs prevents a cell from nucleating MTs Adds to the (-) end No γ- Tubulin= Death 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 (-) end is in the MTOC 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 Gamma-tubulin ring complex plays key role in nucleation MTOC in Dividing Cells Each MTOC has a limited number of nucleation and anchorage sites MT-nucleating activity can be modulated during processes such as mitosis Centrosomes associated with spindle poles in mitotic cells have highest MT- nucleating activity while the spindle is forming MT Stability 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 Proteins involved: o MAPs, microtubule-associated proteins, bind at regular intervals along a microtubule wall, allowing for interaction with other cellular structures and filaments A MAP called Tau causes MTs to form tight bundles in axons Tangles of Tau is related to Alzheimer’s Excess Tau then you will get extensions MAP2 promotes the formation of looser bundles in dendrites o MAPs such as Tau and MAP2 have two regions o One region binds to the MT wall, and another part of the protein extends at right angles to the MT to allow for interaction with other proteins o The length of the extended “arm” controls the spacing of MTs in the bundle Some more Stability Proteins Tip Proteins o MTs can be stabilized by proteins that “capture” and protect the growing plus ends o These are +–TIP proteins (+–end tubulin interacting proteins) o Proteins such as EB1 (end-binding protein 1) decrease the likelihood that MTs will undergo catastrophic subunit loss o Other Depolarizing/Severing Proteins Stathmin/Op18 binds to tubulin heterodimers and prevents their polymerization Catastrophins act at the ends of MTs and promote the peeling of subunits from the ends Proteins such as katanin sever MTs Microfiliments Functions Organization of intracellular organelles and transport of vesicles (motor proteins) Intracellular Motility (e.g. Shigella) Cellular Stability (Stress Fibers) Cellular Motility (Crawling) Muscle Contraction muscle-specific actins (α-actins) Characteristics Smallest (7nm) They are best known for their role in muscle contraction They are involved 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 actinmyocin Microvilli How They are formed 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 G-Actin and F-Actin GTP and ATP 72 nm long for one turn Ropelike Monomer threshold (Kind of Like MT) Need that Critical Concentration to begin formation Polarity of MF Myosin subfragment 1 (S1) can be incubated with microfilaments (MFs) S1 fragments bind and decorate the actin MFs in a distinctive arrowhead pattern Because of this pattern, the plus end of an MF is called the barbed end, and the minus end is called the pointed end 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 hydrolyzed So, the growing MF ends have ATP-actin, whereas most of the MF is composed of ADP-actin Therefore, as you increase the age of MF then you increase the amount of ADP in the MF Again, adds to the plus end more readily Drugs that affect 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 Treadmilling is also found in these guys (you don’t have to know the math!) Structures Commonly FoundWe will talk about these more in depth Structures: Leading Edge (Filopodium) (needed for crawling) o (+) End towards the outside of the cell (adds monomers) o (-) End towards the inside of the cell o microfilaments form highly oriented, polarized cables with the plus ends toward the tip of the protrusion o o Lamellipodium (needed for crawling) o Branched Network of actin o There is order o Gel o There is no order o Stress Fiber Actin Binding Proteins Functions in regulation 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 at the association of MFs into networks Regulation of Monomers and Their Polymerizaiton o If the concentration of ATP-bound G-actin is high, microfilaments will assemble until the G-actin is limiting o In the cell, a large amount of free G-actin is not available because it is bound by thymosin β4 (only binds ATP version) Regulates by not allowing monomers to bind o Profilin competes with thymosin β4 for G-actin binding o ADF/cofilin is known to bind ADP-G-actin and F-actin and is thought to increase turnover of ADP-actin at the minus end of MFs Takes a whole chunk This chunck can be used as a new nucleation site o ADF/cofilin also severs filaments, creating new plus ends in the process Severing of Proteins o MFs are broken up by proteins that sever and/or cap them o Gelsolin breaks actin MFs and caps the newly exposed plus ends, preventing further polymerization Proteins That Cap Actin Filaments o Whether MFs can grow depends on whether their filament ends are capped o Capping proteins bind the ends of a filament to prevent further loss or addition of subunits o CapZ binds to plus ends to prevent addition of subunits there; tropomodulins bind to minus ends, preventing loss of subunits there o Phosphatidylinositol-4,5-bisphosphate (PIP2) can bind to profilin, CapZ, and proteins such as ezrin o PIP2 recruits these proteins to the membrane and regulates their interactions with actin o CapZ binds tightly to PIP2 resulting in its removal from the end of an MF, promoting disassembly Proteins That Crosslink Actin Filaments o Often, actin networks form as loose networks of cross-linked filaments o One of the proteins important in the formation of these networks is filamin o Filamin acts to “splice,” joining two MFs together where they intersect Proteins That Bundle Actin Filaments o Some actin-containing structures can be highly ordered o Actin may be bundled into tightly organized arrays, called focal contacts or focal adhesions o α-Actinin is a protein that is prominent in such structures o Fascin in filopodia keeps the actin tightly bundled o Microvilli Actin bundles in microvilli are the best-studied examples of ordered actin structures Microvilli are prominent features of intestinal mucosal cells; they increase the surface area of the cells The core of a microvillus consists of a tight bundle of microfilaments with the ends pointed toward the tip The MFs are connected to the plasma membrane by crosslinks made of myosin I and calmodulin The MFs in the bundle are tightly bound together by crosslinking proteins fimbrin and villin The Terminal Web o At the base of the microvillus, the MF bundle extends into a network of filaments called the terminal web o The filaments of the terminal web are composed mainly of myosin II and spectrin, which connect the MFs to each other, to proteins in the membrane, and perhaps also to intermediate filaments Proteins that link Actin to membranes MFs are connected to the plasma membrane and exert force on it during cell movement or cytokinesis This (indirect) connection to the membrane requires one or more linking proteins Examples of such proteins include band 4.1, ezrin, radixin, moesin, spectrin, and ankyrin Proteins That Promote Actin Branching and Growth Actin can also form a dendritic (treelike) network A complex of actin-related proteins, the Arp2/3 complex, nucleates new branches on the sides of filaments o causes severe immune deficiency if mutated Arp2/3 branching is activated by a family of proteins that includes WASP (Wiskott-Aldrich syndrome protein) and WAVE/Scar Long Actin Filaments For some cell functions, long actin filaments are needed In this case, actin polymerization is regulated independently of the Arp2/3 complex, through proteins called formins Formins move along the end of the growing filament as they promote polymerization Rho • Both plasma membrane lipids and regulatory proteins affect the formation, stability, and breakdown of MFs • The cytoskeleton of cells exposed to certain growth factors can undergo a dramatic change • Many signals exert their effects by acting on phospholipids, but they also act through a family of monomeric G proteins called Rho GTPases • 3 Classes • Rho pathway-results in formation of stress fibers • Rac- activation results in extension of lamellipodia • Cdc42- activation results in the formation of filopodia Regulation of Rho GTPases Rho GTPases are stimulated by guanine-nucleotide exchange factors (GEFs) through the exchange of bound GDP for GTP GTPase activating proteins (GAPs) inactivate Rho GTPases by causing them to hydrolyze their bound GTPs to GDP Guanine-nucleotide dissociation inhibitors (GDIs) sequester inactive Rho GTPases in the cytosol Intermediate Filaments Characteristics Intermediate filaments (IFs) are not found in cytosol of plant cells but are abundant in many animal cells An abundant intermediate filament (IF) is keratin, an important component of structures that grow from skin in animals IFs are the most stable and least soluble components of the cytoskeleton They likely support the entire cytoskeleton Rope like as well Very Different Chemically inactive Tensile Strength Problems with holding tissue together Can be elastic Intermediate Filaments Confer Mechanical Strength on Tissues Intermediate filaments are thought to play a tension-bearing role In neurons, IFs are dynamically transported and remodeled IFs are less susceptible to chemical attack than are MTs and microfilaments The Cytoskeleton Is a Mechanically Integrated Structure Microtubules resist bending when a cell is compressed Microfilaments serve as contractile elements that generate tension Intermediate filaments are elastic and can withstand tensile forces Intermediate Filament Proteins Are Tissue Specific IFs differ greatly in amino acid composition from tissue to tissue They are grouped into six classes: o Class I: acidic keratins o Class II: basic or neutral keratins o Proteins of classes I and II make up the keratins found in epithelial surfaces covering the body and lining its cavities o Class III: includes vimentin (connective tissue), desmin (muscle cells), and glial fibrillary acidic protein (GFAP) (glial cells) o Class IV: the neurofilament (NF) proteins found in neurofilaments of nerve cells o Class V: includes the nuclear lamins A, B, and C that form a network along the inner surface of the nuclear membrane o Class VI: nestin, the substance that makes up the neurofilaments in nerve cells of embryos o Animal cells can be distinguished based on the types of IF proteins they contain—a technique known as intermediate filament typing Intermediate Filaments Assemble from Fibrous Subunits The fundamental subunits of IF proteins are dimers IF proteins are fibrous rather than globular Each has a homologous central rod like domain of 310 to 318 amino acids in length Flanking the central helical domain are N- and C-terminal domains that differ greatly among IF proteins One Model of Intermediate Filament Assembly The basic structural unit consists of two IF polypeptides intertwined into a coiled-coil The two polypeptides are aligned in parallel Two such dimers align laterally to form a tetrameric protofilament Protofilaments overlap to build up a filamentous structure about eight protofilaments thick Integration of Cytoskeletal Elements Spectraplakins are linker proteins that connect intermediate filaments, microfilaments, and microtubules One, called plectin, is found at sites where intermediate filaments connect to MFs and MTs Cellular Movement and Motility Microfilaments Actin-Based Motility o Crawling Motility o Actin and myosin have been discovered in nearly all eukaryotic cells o They are known to play important roles in nonmuscle motility The Process o Cell Migration via Lamellipodia Involves Cycles of Protrusion, Attachment, Translocation, and Detachment Protrusion Many nonmuscle cells are capable of crawling over a substrate using lamellipodia and/or filopodia Cell crawling involves distinct events: extension of a protrusion, attachment to substrate, and generation of tension, which pulls the cell forward Retrograde Flow: Retrograde flow results from actin assembly at the growing tip of the protrusion and rearward translocation of filaments toward the base Arp2/3-dependent branching drives actin polymerization, particularly in lamellipodia Microtubules are also involved in the process Attachment Attachment, or adhesion, of the cell to the substrate is necessary for cell crawling New sites of attachment are formed at the front of the cell, and contacts at the rear must be broken Attachment sites are complex structures involving a number of proteins Integrins: o Integrins on the outside of cells attach to extracellular matrix proteins o Inside the cell, integrins are connected to actin filaments via linker proteins o The integrin-dependent attachments are called focal adhesions Translocation and Detachment Contraction at the rear of the cell squeezes the cell body forward and releases the attachments at the rear Contraction, due to actin-myosin interactions, is under control of Rho, which activates nonmuscle myosin II at the rear of the cell For movement to occur, new attachments must be balanced by loss of old ones o Motility vs Contractility Motility Movement of a cell or organism through the environment Movement of the environment past or through a cell Movement of components in the cell Motility occurs at the tissue, cellular, and subcellular levels Intracellular components move; for example, microtubules of the mitotic spindle play a role in the separation of chromosomes during cell division To generate movement, microtubules (MTs) and microfilaments (MFs) provide a scaffold for motor proteins that produce motion at the molecular level 2 Types of Systems from Eukaryotes o Microtubule-based motility Examples: fast axonal transport in neurons; the sliding of MTs in cilia and flagella o Microfiliment-Based motility Muscle Contraction Contractility used to describe shortening of muscle cells, is a specialized form of motility o All about the engine that drives them along They couple ATP hydrolysis to changes in shape and attachment of the motor protein They undergo cycles of ATP hydrolysis, ADP release, and acquisition of new ATP They have common structural features They can move along a cytoskeletal filament for significant distances Microtubules Motors o Kinesins (mostly + end) anterograde Kinesins consist of two dimerized heavy chains and two light chains The heavy chains contain globular domains that attach to microtubules, a coiled-coil stalk, a lever-like neck that connects the two, and a tail The light chains are associated with the tail Kinesins are classified into families based on their amino acid sequence Some form homodimers; others, heterodimers Kinesin-14 is minus-end directed; kinesin-5 is bidirectional The kinesin-13 family, the catastrophins, aid depolymerization of MTs. Movement along MT Kinesin movement looks like “walking,” with the two globular head domains taking turns as the front foot Each kinesin molecule exhibits processivity It can move long distances along an MT before detaching from it by releasing bound ADP and acquiring a new ATP, so that the cycle repeats 1 ATP=1 step=8nm o Dynein (mostly – end) retrograde (The drunken sailor walk) So think of Dyneins as the drunken sailor pulling a trailer. The Dynactin is the bed of the trailer and acts as Velcro for the vesicle to sit on. o o Movement inside cells o MTs provide a rigid set of tracks for transport of a variety of organelles and vesicles o Traffic toward the minus ends of MTs is considered “inbound” (dyneins); toward the plus end is “outbound” (kinesins) o Microtubule-associated motor proteins—kinesins and dyneins—walk along the MTs and provide the force needed for movement Axonal Transport Proteins produced in the cell body are transported to the nerve ending in a process called fast axonal transport This involves packaging of these proteins into vesicles for transport Organelles can also be observed moving along filaments through axoplasm (cytoplasm of axons) at rates of about 2 μm/sec ( So, for a vesicle to travel down the axon from the spinal cord to the foot, it would take about 5 days! (This is going really fast even through it does not seem like it. Proteins responsible o Kinesin I is involved in ATP-dependent transport toward the plus ends (away from the centrosome), called anterograde axonal transport o Cytoplasmic dynein moves particles (cargo) in the opposite direction, called retrograde axonal transport