Cytoskeleton

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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