cytoskeleton
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Lecture Handouts in Cellular and Molecular BiologyTRANSCRIPT
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The Cytoskeleton and Cell Motility
Cell and Molecular BiologyLecture 9
John Donnie A. Ramos, Ph.D.Department of Biological SciencesCollege of Science University of Santo Tomas
Cytoskeleton
The “skeletal system” of cells
Composed of filamentous structuresMicrotubules – rigid tubes
tubulin
Microfilaments – solid, thin structuresactin
Intermediate filaments – tough, ropelike fiberskeratin, vimentin, desmin, Lamin etc.
Highly dynamic structures
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Functions of the Cytoskeleton
Microtubules and Peroxisomes
Peroxisomes are labeled with GFP while microtubules are labeled with antibodies fused with a red fluorescent dye.
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Studying the Cytoskeleton
Fluorescence microscopyReal-time study
Ideal for studying the dynamics of cytoskeleton
Uses antibodies and fluorescent dyes/proteins
Identifying the location proteins in small amounts
centrin
Studying the Cytoskeleton
Video microscopyMonitoring of cell movement (real-time)In vitro motility assayUsed to study motor proteinsMade possible with the development of nanotechnology
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Studying the Cytoskeleton
Genetically engineered cellsBased on recombinant DNA technologyUses modified proteins (site directed mutagenesis)Knockout animalsOverexpression of mutant proteins (transfection)
Normal pigment cell with pigment granules equally distributed throughout of
the cell with the help of kinesin II
Mutant cell conatining modified kinesin II resulting in aggregation of pigment granules
Microtubules
Hollow, tubular structuresFound in nearly all eukaryotic cellForm mitotic spindle, flagella, ciliaOuter diameter: 24 nm; thickness: 5 nmComposed of 13 protofilaments (globular proteins)
Plus (+) end
Minus (-) end
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Microtubule-Associated Proteins
MAPs are found only in brain tissue
With a domain attached to microtubule and a domain extending outward as a filament
Form cross bridges connecting microtubules
Alter rigidity or influence the rate of assembly
Phosphorylated proteins
Mutation of a MAP protein (tau) resulting to overphosphorylationcauses dementia (formation of neurofibrillar tangles)
Microtubule Function
Structural Support and OrganizersServe as mechanical supportDetermine cell shapeTracks for organellesMaintains internal organization of cells
Microtubules of culture mouse cell
Axon
VesiclesLysosome
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Microtubule Function
Agents of intracellular motilityAxonal transport (movement of proteins and neurotransmitters along the axon) Rate of movement: 5 µm/sec (400 mm/day)Both anterograde and retrograde directionMicrotubules are the main tracks
Motor Proteins
Utilizes ATP – conversion of chemical energy to mechanical energyTypes of motor proteins:
Myosins – move along microfilamentsKinesins – move along microtubulesDyneins – move along microtubules
Move in unidirectional manner along a trackDuring movement, motor proteins undergo mechanical and chemical cycle (series of conformational changes providing the necessary fuel for movement)Steps:
ATP binding to motor proteinATP hydrolysisRelease of products (ADP and Pi)
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Kinesins
First isolated in 1985 from squid giant axonsTetramer (2 identical heavy chains and 2 identical light chains)Globular head – force generating “engine”Tail region – binds to cargo (vesicle)Movement is a coordinated activity between the 2 heads)Movement towards + endBelong to a family of proteins called KLPs (Kinesin-like Proteins) or KRPs(Kinesin-related Proteins)XKCM1 – KLP that is incapable of movement (destabilize microtubules)
Kinesin-Mediated Organelle Transport
Microtubules
Mitochondria
Normal 9.5 day mouse embryo cell
KIF5B kinesin-deficient 9.5 day mouse embryo cell
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Cytoplasmic Dynein
First isolated in 1963 from cilia and flagella (cytoplasmic form was discovered in 1983)Molecular mass: 1.5 million D2 identical heavy chain and variety of intermediate and light chainsMovement towards minus endFunctions:
Spindle positioning and chromosome movement during cell divisionMinus end-directed positioning of GCMovement of vesicles and organelles in the cytoplasm
Dynactin – regulate dynein activity and binds dynein to microtubules
Model of Motor-Mediated Transport
Kinesin: anterograde
movement
Dynein: retrograde and anterogrademovement
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Microtubule-Organizing Centers
Specialized structures involved in microtubule nucleation and organizationMicrotubules – assembly of αβ-tubulin dimersStages of microtubule assembly:
Nucleation – slower phase (initial formation of a part of microtubule)Elongation – rapid phase (formation of the entire organelle)
CentrosomesA complex of 2 centrioles surrounded by amorphous, electron dense pericentriolar material (PCM)Site of microtubule nucleationCentrioles
9 triplets of tubules In pairs arranged at right angle
Minus end of microtubule is associated with centrosomewhile plus end (growing end) is situated on the other side.
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MTOC of Plant Cells
Plant cells lack both centrosomes and centriolesMicrotubule-organizing center originates near the nuclear envelopeMicrotubule nucleation throughout the plant cell cortex
Localization of microtubules Origin of nucleation on the nuclear envelope
Microtubule Nucleation
Tubulin – protein component of all MTOCsTypes of tubulin:
γ tubulinthe main protein involved in microtubule nucleation0.005 % of the total cell protein content
β tubulinα tubulin
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Dynamic Nature of Microtubules
Microtubules assemble and disassemble regularlyResult of polymerization and depolymerization
Interphase:microtubules
distributed throughout the cell cortex
Pre-prophase:microtubules form a single
transverse band (preprophase band)- site of future division plane
Mitosis: microtubules form spindle fibers (mitotic spindles)
Late Telophase:microtubules form
phragmoplast (involved in cell wall formation
between daughter cells)
Microtubules Assemble In Vitro
First performed by Richard Weisenberg in 1972 using brain cell homogenate.Assembly occurs at 37°C but disassembles at lower or higher temperaturesRequires GTP for assembly (after binding of tubulin dimer to a growing microtubule)GTP is hydrolyzed to GDPStructure cap model of dynamic instability (a process of assembling and disassembling microtubules)
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Microtubules Assemble In Vivo
Dynamic Instability -ability to grow and
shrink (assemble and disassemble)
in vivo
Eukaryotic Cilia
Locomotory organsContains microtubules with dynein armsResponsible for basic vertebrate body plan (sidedness of some organs)Cells of the embryonic node during gastrulation stage contain cilia responsible for the movement of cellsAbnormal cilia causes “situs inversus”
Protozoan cilia in metachronal wavesCilia on the surface of the fimbrium of mouse oviduct
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Eukaryotic Flagella
Locomotory organsContains microtubules with dyneinarmsFewer but longer (compared to cilia)
Chlamydomonas reinhardtii
“Breast stroke”-like movement
Ciliary or Flagellar Axoneme
Axoneme is the central core of cilia or flagella containing 9 doublets and 1 central pair of microtubules (9+2 array structure). Microtubules are of same polarity (-end at the base and + end at the tip)
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Ciliary or Flagellar Axoneme
Longitudinal view of axoneme Basal bodies and axoneme
Ciliary Dynein
Protein responsible for the conversion of ATP into mechanical energy of ciliary locomotion
Discovered by Ian Gibbons using an experiment involving the “chemical dissection of cilia from the protozoan Tetrahymena
Heavy chains
Intermediate chains
Light chains
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Ciliary Motility
Causes motility by sliding mechanism (microtubule sliding)Dynein arms act as swinging cross-bridges between A and B tubules
Intermediate FilamentsSize between microtubules and microfilamentsSolid, smooth-surfaced, unbranchedAverage diameter: 10 nmFound around cytoplasmHeterogenous group of cytoskeletons encoded by at least 50 different genes in humansBasic unit pattern: tetramer (antiparallel, staggered dimers)Tetramer lacks polarityDimers exist as homodimers or heterodimers
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Major Mammalian IFs
Keratins
Type I keratinsAcidic keratins
Type II keratinsBasic or neutral keratins
Keratin heterodimers are combinations of different typesOriginate on the nuclear membrane, radiate around the cytoplasm and terminate in desmosomesand hemidesmosomes
Cultured skin cells (keratinocytes)
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Neurofilaments
Fibers are parallel to nerve cell axon
Type IV proteins: NF-L, NF-H, NF-M
Main supporting cytoskeleton of neurons (axons) as they increase in diameter
MicrofilamentsMajor contractile proteins of muscle cellsComposed of actinAlso called “actin filament” or “F actin”8 nm in diameter (smallest among cytoskeletons)Responsible for cell and organelle motilityActin monomer composed three subunits (each subunit with 4 subdomains)Can form double helical structure (dimer)Highly conserved geneInteracts with myosin (motor protein)
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Actin Assembly
Actin is an ATPase (binds ATP)
Polymerization and depolymerization of actin filaments can be simulated in vitro
Addition (assembly) of actin subunits occurs at the + end (fast growing end)
Disassembly occurs at the – end
Microfilament assembly and disassembly can be inhibited by cytochalasins (promotes depolymerization of microfilaments) and phalloidin ( increase microfilament stability)
Actin Polymerization
Force-generating mechanism for cell motility without the use of motor proteins
Examples:Acrosomal Reaction (rapid extension of the acrosomal filament to the antierior tip of spermatozoon)
Propulsion of of Listeriamonocytogenes to the cytoplasm of an infected cell
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Myosin
Molecular motor of actinfilaments
2 globular headsActin binding site
Catalytic site (hydrolyses ATP)
2 neck regions (α helix) – heavy chain
2 essential light chains
2 regulatory light chains
Tail region – intertwined heavy chains (coiled coil protein)
In Vitro Motility Assay for Myosin
Myosin heads immobilized on cover slip
Allowed to react with actin extracts
Observed for movement of actin
Video microcopy
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Bipolar Myosin II Filament
Myosin heads (globular) on opposite ends while tails overlap at the center of the filament
Plays a structural role in muscle cells (stable componant of the contractile apparatus)
Results in a bipolar filament
Uncoventional Myosins
Myosin IWith single headCannot assemble into filaments (in vitro)Can move actin (motor protein)Localized on plasma membrane (generates forces on cell surface)First isolated in Acanthamoeba
Myosin VInvolved in the transport of pigment cells in humansAbnormality results in partial albisms
Myosin VIILocallized in hair cells of cochlea of the inner earMutation results deafness
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Muscle Fibers
Results of myoblast fusion (multinucleated)10-100 µm thickComposed of myofibrils with contractile units called sarcomeresSarcomere composed of overlapping actin (thin) and myosin (thick) filaments Each sarcomere separated by a Z line
Contractile Machinery of Sarcomere
M Line
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Sliding Filament Model
Explains the mechanism of muscle contractionDecreased width of the I bandand H zone
Associated Proteins
TropomyosinRod-shaped protein associated with 7actin filaments
TroponinBinding site of Ca+ during muscle contaction
TitinPrevents overstretching of sarcomere
NebulinRegulates the number of actin monomers assembling into thin filaments
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Actinomyosin Contractile Cycle
1. ATP-binding (actindetaches from myosin)
2. Hydrolysis of ATP3. Weak interaction of
actin with myosin4. Release of Pi (Power
Stroke)5. Release of ADP
(attachment of myosin to actin)
Functional Anatomy of Muscle Fiber
1. Arrival of nerve impulse causes the release of Ca+ from SR
2. Ca+ binds to troponin3. Conformational change of
troponin subunit4. Movement of trpomyosin5. Myosin binding site is
exposed thus myosin interacts with actin
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Actin-Binding Proteins
Actin-Binding Proteins
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Nonmuscle Motility and Contractility
CytokinesisPhagocytosisCytoplasmic streamingVesicle traffickingBlood platelet activationLateral movement of integral membrane proteinsCell-substratum interactionsCell locomotionAxonal outgrowthsChanges in cell shape
Stress fibers