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The cytoskeleton Function, design and regulationCell Biology interactive media video or animation1

Chapter 16965-10201026-1050A lot of reading!Focus on general principlesand topics highlighted inthe lecture synopsis Lecture 8 & 9:Cell biology 2014 (revised 11/2-14)1Classical cytoskeletons Actin filaments(Microfilaments) Intermediate filamentsMicrotubules

21. Why do we need a cytoskeleton?Cell containing cytoskeleton

Cell without cytoskeleton

ER Golgi

Establishment of cellular shape and intracellular organization Resistance against mechanical stress1132. Why do we need a cytoskeleton?Cell containing cytoskeleton

Cell without cytoskeleton

Cellular appendages Genomic and cellular division Cell locomotion4Principle architecture of cytoskeletal filaments25 nmMicrotubulesSubunits:7 nmActin filamentsActinTubulin heterodimer10 nm Intermediate filamentsA family of coiled-coil proteins5Non-polar filaments

Cytosol: support of cell layers ( tensile stress )

Nucleus: supporting the nuclear envelopeIntermediate filaments structure and functionTetramer of coiled-coil dimersamphipathic a-helical monomers+6Cell adhesion(desmosome)

animation 16.4-Intermediate _filamentProtein:

Location:CytosolicNuclearCyto-keratinsVimentin, DesminNeuro-filamentsLaminsLining of the nuclear membrane of all cellsEpitheliaNeuronsCells in connective- and muscle tissueTissue specific intermediate filamentsIntermediate filaments can be composed of either: Homodimers or heterodimers - Intermediate filament super-family: >60 genes in mammals7Intermediate filaments in epidermis 1 + 105 + 14PPPPPPPPKeratin:

PPPPPPPPPPPPPPPP8ECM(Basal lamina)8

Actin filaments structure and functionStructure - Polar filaments composed of actin

Functions - Linking the interior to the exterior ( )

- Contraction ( )

- Spreading & protrusions cell shape

- Locomotion

- Contractile ring during cell division Video 01.1-keratocyte_dance9Video 22.7 neurite_outgrowthECM9Actin filaments are dynamic in migrating cellsTimeCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaStimuliRapid assembly and disassembly is central to a variety of functions, such as cell remodeling and locomotion Coca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca Cola10Coca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaA polymer with only longitudinal subunit interactions uniform (and poor) polymer stability+A polymer with both longitudinal and lateral subunit interactions stability within the polymer but dynamic ends Coca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaDynamic endsCoca ColaCoca ColaCoca ColaCoca ColaInternal stability+Coca ColaCoca Cola11Subunit interactions within actin filamentsProtofilament(proto = a prefix meaning the earliest)Nucleation of actin filamentsSpontaneous nucleation is slow because the initial interactions are unstable (low degree of cooperativity)Coca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaSpontaneous nucleationFantaFantaSpatially regulated nucleation factors local nucleationNucleation factorCoca ColaCoca ColaFantaFantaCoca ColaFantaFantaCoca ColaCoca ColaCoca ColaCoca Cola12FantaFantaControl of actin filament nucleationNo availabilenucleation factor Inactivenucleation factorCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaFantaFantaFantaFantaFantaFantaCoca ColaFantaFantaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaFantaFantaFantaFantaCoca ColaCoca ColaCoca ColaFantaFantaCoca ColaCoca ColaCoca ColaFantaFantaCoca ColaCoca ColaCoca ColaFantaFantaCoca ColaCoca ColaCoca ColaGlobal activationof nucleation factorsNo (specific) nucleationGlobal nucleationLocalized activationof nucleationfactorsLocal nucleation13No (specific) nucleationCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaActin nucleation factorsArp 2/3 Arp 2/3 may alsobind pre-existing filaments to createbranching+ end- endFormin+ end- end14Coca ColaCoca Cola Concept of the critical free concentration% Subunits in filament(% Bound) Time (minutes)Coca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaSpontaneousnucleationElongationCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaSteady stateCoca ColaThe monomer ( = soluble subunit) concentration ( = [Free]) at steady state is referred to as the critical concentration 100% Coca Cola15 Monomer concentration [Free subunits] 00 %60Coca Cola Dynamics at different free concentrations [Monomers] < critical concentration, e.g. 1 nMCoca ColaCoca ColaNet depolymerization16Coca Cola [Monomers] > critical concentration, e.g. 3 nMCoca ColaCoca ColaNet polymerizationCoca ColaCoca ColaCoca Cola [Monomers] = critical concentration, e.g. 2 nM

Coca ColaCoca ColaNo net effect on polymer lengthCoca ColaCoca Cola[Monomer] = [Free subunits] Coca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca Cola Nucleotide turnover in cytoskeletal subunitsThe subunit changes its conformation upon nucleotide hydrolysisCoca ColaCoca ColaSubunit bound to a nucleosidetriphosphateActin subunits bind ATPTubulin heterodimers bind GTPNucleotide hydrolysisSubunit bound to a nucleosidediphosphateA nucleoside is the portion of a nucleotide that doesn't include the phosphate groups17Treadmilling occurs when [Monomers] (i.e. [Free subunits]) is between the critical concentrations at the two ends (2 10 nM)High critical concentration(e.g. 10 nM)Low criticalconcentration(e.g. 2 nM)+ end- endDuring treadmilling the filament length remains constant, while subunits are added at the (+) end and dissociate from the (-) end18Coca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaCoca ColaATPADPI. ATP fueled actin treadmillingII. ATP fueled actin treadmilling:interaction strength+-19Treadmilling actin subunits "move" towards the (-) end+-ATPADPPADPPADPADPATPADPPATPATPADPADPPADPADPADP[ADP]