History of Cell Biology:

1. Invention of Microscope:
• Zacharias Jansen:
• First compound microscope.
• Magni cation 9X.
• Hooke:
• Magni cation 50X.
• Di erent ways to shine light & magnify specimens.
• Described cells for the rst time.
• Leeuwenhoek:
• Magni cation 230X.
• Used better lenses.
• Reinhold Rüdenberg:
• Patented electron microscope.
• Ruska & Knoll:
• Invented and built the rst electron microscope.
• Xiaowei Zhuang:
• Invented Stochastic Optical Reconstruction Microscopy (STORM)
• Eric Betzig:
• Invented Photo-activated Localization Microscopy (PALM)
2. Cell Theory:
• All living organisms are composed of one or more cells.
• The cell is the basic unit of structure and organization in organisms.
• Cells arise from pre-existing cells.
3. Development in Cell Biology:
• Robert Remak:
• Found a way to harden and stain cells.
• Found cell membrane.
• Rudolf Virchow:
• Stole the idea from Remak and got credit for Cell Theory.
• Albert von Kölliker:
• Discovered mitochondria.
• Carl Benda:
• First used the term “mitochondria”.
• mitos = thread; chondros = granule.
• Camillio Golgi:
• Invented the Dark Reaction (Golgi stain).
• Discovered the Golgi apparatus.
• George Otto Grey:
• Made the rst cell line (HeLa cells).
• Rudolph Jaenisch:
• First transgenic mice but DNA did not get into germline.
• Osamu Shimomura:
• Identi ed Green Fluorescent Protein from jelly sh.
• Douglas C. Prasher:
• Cloned GFP gene and proposed it could be used as molecular tracer.
• Roger Tsien:
• Identi ed mutants of GFP with enhanced spectral properties.
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 Microscopy:
• 20th century:
• Better lenses, better light sources.
• Use electricity to excite mercury vapour —> generate light.
• Objective lenses with di erent magni cations on a turret —> can quickly change
magni cation.
• Limit of resolution = 0.2 μm
• Modern compound microscopes:
• Light path.
• Dichroic mirrors:
• Allow light of a certain wavelength to pass through.
• Re ect all other wavelengths.
• Magni cation:
• Objective lens: max 100X
• Projection (eyepiece) lens: 10X
• Light microscopy:
• Bright eld:
• Unstained samples.
• Most basic.
• 1 light source —> concentrated onto the sample by condenser lens.
• Phase-contrast:
• Unstained samples.
• Utilizes refractive index of the sample.
• Light moves slower in areas with higher refractive index.
• Produces images with dark and light areas based on refractive index of these zones.
• Annular diaphragm & phase plate:
• Only allows some of the light to pass through.
• Di erential interference contrast (DIC):
• Unstained samples.
• Breaks light into 2 perpendicular components —> hit sample —> light recombined.
• Observe interference pattern.
• Useful for seeing small details.
• Generate shadow-like looks.
• Fluorescence microscopy:
• Excitation lter: only allows a certain wavelength of light to pass through.
• Dichroic mirror: re ects certain wavelengths —> hits sample labeled with
uorochrome—> uorochrome emits uorescence.
• Immuno uorescence:
• Secondary antibody attached with a uorochrome.
• Secondary antibody binds primary antibody.
• Fluorescence allows localization of the primary antibody.
• Molecular probe:
• Molecule A attached with a uorochrome.
• Molecule A binds Molecule B.
• Fluorescence allows localization of Molecule B.
• Problems:
• Light excites the entire sample, but we only want to focus on one plane of the
sample to image.
• Phototoxicity: high-powered light sources create free radicals —> kill cells.
• Confocal microscopy:
• Uses lasers —> produces sharp image with little out-of-focus light.
• Problem: laser is intense —> uorochromes can bleach out (phototoxicity
throughout the specimen even though light is only collected at one focal plane)
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 • Confocal point-scanning microscopy:
• Takes image from only 1 speci c focal plane.
• Very little out-of-focus light
• Collects images from entire sample —> reconstruction to create 3D model.
• Pinhole: further ensures that out-of-focus light is eliminated.
• Problems: slow & not suitable for live imaging.
• Confocal spinning-disc uorescence microscopy:
• Splits laser by many lenses and pinholes.
• Scan entire sample in milliseconds.
• Decreases exposure time and intensity of laser beam.
• Used for live imaging.
• Multi-photon uorescence microscopy:
• Uses multiple lasers at 1/2 or 1/3 or 1/4 of their intensity.
• Same emission from the uorochrome but less phototoxicity.
• Ideal for live imaging.
• Deconvolution:
• Get confocal image quality with a regular uorescent microscope.
• Uses software calibrated to the microscope system.
• Image uorescent beads with known sizes as controls.
• Mathematically move out-of-focus light back into focus.
• Fluorescence recovery after photobleaching (FRAP):
• Study protein turn-over.
• A region of a uorescently-tagged protein is hit with a laser for a prolonged period
until the uorochromes in that area is bleached.
• Fluorescence will recover —> recover time tells us the turnover rates of proteins.
• Fluorescent resonance energy transfer (FRET):
• See if 2 molecules are close together.
• Uses 2 uorescent proteins —> excite the rst one —> emits light in a
wavelength that will excite the 2nd one if they are close —> collect light from the
2nd molecule.
• Super resolution microscopy:
• Allows to get past the optical resolution limit of 0.2 μm.
• Structured illumination (SIM):
• Places a grid between light path and camera —> grid is rotated.
• 3 pictures are taken with the grid in 3 di erent positions.
• Only the light in the same position is kept —> sharp image.
• Resolution 100 nm.
• Problem: very slow.
• Stimulated emission depletion (STED):
• Has a depletion beam around the laser point —> much smaller excited area.
• Reconstruction using all of the points.
• Resolution 30 nm.
• Problem: very slow.
• Photo-activated localization microscopy (PALM):
• Uses photo-activatable uorescent proteins —> black to start with, uoresce
when activated.
• Thousands of images are collected when only a few are excited.
• Centre of the excited uorescent protein is documented, collected, and used
to reconstruct an image.
• Resolution 10 nm.
• Problem: very very very slow.
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 • Stochastic optical reconstruction microscopy (STORM):
• Uses photo-switchable uorochrome —> excite to a dark state.
• Centre of each molecular image is plotted based on photons emitted.
• Resolution 5 nm.
• Lattice light-sheet microscopy:
• Uses a sheet of light to illuminate 1 plane of the sample.
• All planes are imaged very quickly.
• Resolution 150 nm.
• Live super-resolution imaging.
• Thick samples:
• Problems:
• Image quality get progressively worse as thickness increases.
• Have to use infrared wavelengths of light for thick samples —> GFP is not in IR
spectrum.
• Solution — tissue clearing:
• Current techniques can preserve membranes —> but must clear lipids.
• Hydrogel embedding:
1. Embed samples in hydrogel (meshwork to hold tissue in place).
2. Remove lipids in 8% SDS.
3. Immerse in clearing solution.
4. End up with clear sample.
• May cause the tissue to shrink or swell.
• No membrane hampering the permeability of molecular probes.
• Can take hours to months.
• Electron microscopy:
• Uses high velocity beam of electrons to shoot at a sample.
• Resolution 0.0005 nm due to short wavelength of electrons.
• Can’t do live imaging.
• Must be under ultrahigh vacuum.
• Transmission electron microscopy:
• Samples xed by cross-linking proteins with xatives.
• Embedded in a resin & cut into 70 nm sections.
• Sample is stained with heavy metals —> gives contrast.
• Dark: electron hit the metal.
• Light: electron did not hit the metal.
• Scanning electron microscopy:
• Samples are coated with metals.
• Used to see the surface of samples.
• Image = secondary electrons released from the metal & detected using computers or
cathode tube.
• Resolution 10 nm.
• CryoEM:
• Used to identify protein structures.
• Samples frozen with liquid nitrogen.
• Samples viewed in native state.
• Problems with modern electron microscopy & uorescent microscopy:
• Can’t do it both with the same sample.
• miniSOG:
• Small genetic tag for proteins (106 amino acids)
• Fluorescent like GFP
• Can catalyze reaction of diaminobenzidine —> reaction product can be stained with
osmium (heavy metal).
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 Actin:
• 15% of all proteins in eukaryotic cell.
• Highly conserved among all species.
• Functions:
• Generating/maintaining cell structure.
• Adhesion to substrates.
• Cell division.
• Intracellular motility.
• Whole cell motility.
• G-actin:
• Globular actin.
• Monomeric form.
• Have 2 lobes that house the Mg-ATP at the centre.
• F-actin:
• Filamentous actin.
• Polar —> have directionality —> can be used as tracks.
• Formed from incorporation of G-actin onto the ends —> powered by ATP hydrolysis.
• Barbed (+) end grows faster than (-) end.
• Nucleation:
• Initiation of the polymerization process.
• Spontaneous nucleation at a site with an actin-trimer —> unfavorable con rmation —.
usually dealt with by using actin associated proteins.
• Nuclear actin:
• Both monomeric & lamentous form.
• Regulates gene transcription by regulating:
• Transcription factors (eg. MKL1):
• MKL1 = coactivator of SRF —> involved in cell motility.
• G-actin binds and inactivates MKL1 —> SRF not expressed.
• RNA Pol:
• G-actin can bind directly to RNA Pols and P-TEFb.
• Chromatin regulating complexes:
• When double-stranded breaks occur in cells to remove DNA segments, F-actin is
used to drag the segments away to prevent recombination (by using the motor
myosin).
• How does G-actin get in and out of nucleus?
• Importin-9: get G-actin into the nucleus.
• Exportin-6: get G-actin out.
• Both activated by G-protein Ran.
• Actin-associated proteins:
• Pro lin:
• Binds to G-actin & catalyzes exchange of ADP to ATP.
• Binds to polyproline residues in other actin-associated proteins.
• Adds G-actin monomers to barbed end of actin laments.
• Inhibits the spontaneous nucleation
• Co lin:
• Destabilizes and severs ADP-actin laments.
• Leads to disassembly of actin laments.
• Drags ADP-actin monomer out of the lament —> can start nucleation if the actin can
form a trimer with 2 other actin monomers.
• LIM kinases (LIMK) 1 & 2:
• Phosphorylates co lin on Ser-3.
• Blocks co lin-actin interaction.
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 • Stabilizes actin laments.
• Activated by GTPases (Rac, Rho, Cdc42).
• Dephosphorylated by Slingshot.
• Slingshot:
• Dephosphorylates & activates co lin.
• Destabilizes actin laments.
• Can also dephosphorylate LIMK.
• Gelsolin:
• 6-domain protein.
• Binds to the sides of actin laments & wraps around it.
• Severs & caps actin laments.
• When it severs the lament it dose not let go —> stays attached to the end of the
lament.
• Activated by micromolar concentrations of Ca2+.
• Capping protein (CapZ):
• Heterodimer of α & β subunits.
• Binds to free barbed end.
• Cap actin laments & block addition of actin monomers to barbed end.
• Promote nucleation of new actin laments by stabilizing small actin oligomers at
pointed end.
• Arp2/3 complex:
• Actin nucleator.
• Associated with 5 other proteins.
• Binds to pre-existing actin laments & nucleates new branches.
• Caps & nucleates pointed ends of new laments (pointed end = root)
• New lament grows from barbed end.
• Resulting in branched actin laments.
• Arp2 & 3 structurally resemble actin.
• Act as pseudodimers.
• Addition of 1 G-actin monomer can lead to spontaneous nucleation.
• Usually inactive —> activated by WASP.
• WASP:
• Nucleation promoting factor
• Most cells contain N-WASP.
• WH1 (EVH1) domain:
• EVH1 = Ena/Vasp-homology-1.
• WH1 = WASP-homology-1.
• Binds to proline-rich region of WASP-binding proteins.
• Basic domain:
• Binds to PIP2.
• Allows WASP to be anchored to plasma membrane.
• GBD:
• GTPase-binding domain.
• Binds to Cdc42.
• Proline-rich domain:
• Binds to SH3 domain of other proteins.
• V domain:
• Binds 1 actin monomer.
• Brings the actin monomer to Arp2/3 complex to start nucleation.
• C (co lin) & A (acidic) domains:
• Bind Arp2/3 complex.
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 • VCA domain together activates Arp2/3 by causing conformational change —> Arp2 &
Arp3 are spaced out to nucleate actin.
• Auto-inhibited when not in use.
• Folds back on itself through interactions between Basic, GBD & VCA domains.
• Activated by Cdc42.
• Binds to GBD.
• Causes WASP to open & interact with actin & Arp2/3
• Ena/Vasp proteins:
• Stimulates actin lament elongation & decrease Arp2/3-based branching.
• Protect laments from capping.
• Binds barbed end.
• Enhances delivery of pro lin-actin to barbed ends.
• Increases speed of actin-based movements.
• WAVE:
• WH2 domain.
• 20 amino acids found in the V domain.
• Binds actin monomer.
• Also has VCA domain to activate Arp2/3.
• Part of a larger complex with 4 other proteins.
• If any of the components are not present, the complex breaks apart and is
degraded.
• Not auto-inhibited.
• Activated by Rac.
• Arpin:
• Arp2/3 inhibitor.
• Localizes to lamellipodia.
• Activated by Rac.
• Has the A domain but lacks V & C —> binds Arp2/3 but does not activate it.
• Arpin present —> Arp2/3 blocked —> cell directionality changes.
• Spire:
• Monomer but can dimerize.
• 4 WH2 domains.
• Short linker between WH2-3 & -4.
• Close association stabilizes actin laments.
• KIND domain interacts with Formins.
• Dimerizes upon interaction with Formin —> 8 WH2 domains —> can polymerize 2
actin strands.
• Nucleates actin very slowly.
• Cordon-bleu:
• 3 WH2 domains.
• Long linker between 2nd and 3rd WH2.
• Formins:
• Elongate actin laments that are straight —> not networked or branched.
• Remain associated with barbed end —> prevent capping.
• All have FH2 domain.
• Doughnut shape around barbed end.
• 1 FH2 domain + 2 actin monomers = spontaneous nucleation.
• FH1 domain:
• Proline-rich —> allow docking of pro lin-ATP-G-actin.
• Activated by GTP-Rho.
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 • Actin-based structures:
• Lamellipodia:
• Leading edge of whole-cell movement.
• Structures made of membrane & WASP-Arp2/3 branched actin.
• Actin polymerization occurs at membrane surface.
• Filaments wiggle & vibrate to give space for incorporation of more actin
monomers beneath the membrane.
• Filopodia & microspikes:
• Sample the cellular environment and direct cell movement by guiding lamellum
protrusion.
• Actin arranged in parallel arrays —> highly cross-linked by actin-bundling proteins.
• Microspikes = have not yet protruded out —> still within the lamellipodia.
• Filopodia = emerge out of the lamellipodia.
• Microvilli:
• Increase membrane surface area for absorption.
• Actin-dense cores of 20-40 parallel and cross-linked actin laments.
• Barbed ends up towards cell periphery.
• Stress bres:
• Parallel bundles of 10-30 laments —> highly cross-linked.
• Actin laments themselves run in anti-parallel fashion —> required for contraction.
• Never seen in vivo.
• Most are contractile —> have myosin.
• Actin-bundling proteins:
• Used to cross-link and bundle parallel actin laments.
• Fascin:
• Found in parallel actin structures at actin periphery (microspikes & lopodia).
• Controlled through phosphorylation.
• Phosphorylation by PKC —> loss of actin bundling.
• Rac can act on Fascin-PKC complex and reverse the phosphorylation/
inactivation.
• Upregulated in many di erent cancers.
• Fimbrin:
• 2 actin binding sites.
• Completely lls up actin core in microvilli.
• Espin:
• 20 actin binding sites.
• Found in Ectoplasmic Specializations of testes, microvilli, hair cells, etc.
• L-Espin & S-Espin:
• Mutations lead to deafness.
• Have WH2 domains —> bind actin.
• Can bind pro lin and elongate actin.
• Villin:
• 3 actin binding sites.
• 2 are Ca2+-dependent.
• Member of the gelsolin family.
• Can bundle, cap, sever, nucleate actin laments.
• Infection by bacteria Shigella requires villin.
• α-actinin:
• 2 actin binding sites.
• Member of the spectrin family.
• Forms antiparallel rod-shaped dimers with 1 actin-binding site at each head.
• Spectrin repeats can resist molecular mechanical strain.
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 • Actin & Actin-associated protein drugs:
Drug Name Target Origin Binding site Function

Barbed-end
Cytochalasin Fungi
groove. Inhibits actin
Actin monomer
monomers Nucleotide-binding incorporation into
Latrunculin Sponges actin lament.
cleft.

• Met 119, 355, Glu

• Stabilizes actin
117
laments.
• Binding site only
• Prevents actin
Amanita exposed & in
Phalloidin Actin disassembly.
mushrooms proper
laments • Can be used to
conformation
label actin laments
when actin is in
in xed cells.
lamentous state

Only to actin Inhibits actin lament

Jasplakinolide Sponges
laments disassembly

• Blocks
conformational
change needed to
active Arp2/3.
CK-666 Arp2/3
• Inhibits branched
actin formation.
• Reversible if
removed.

• Inhibits nucleation
and elongation of
SMIFH2 Formin actin laments.
• Decreases formin-
barbed-end a nity.
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 Microtubules:
• Assembly:
• Formed by α & β tubulin heterodimers.
• Both monomers associates with GTP.
• α-tubulin-GTP remains bound and locked between α & β monomers.
• β-tubulin-GTP is rapidly hydrolyzed to GDP during/after polymerization —>
destabilizes interaction with adjacent molecules —> dynamic instability.
• β-tubulin-GTP is stable at microtubule pole, but unstable within the core —> GTP cap
locks tubulin in a straight conformation.
• 13 proto laments form a sheet in mammals.
• Doublecortin: protein found at the (+) end of microtubules.
• Shape of 13 proto laments enables binding of doublecortin.
• Stabilizes microtubule.
• Mutation in microtubule-binding domain leads to lissencephaly.
• Proto lament sheet rolls up and seals into a tube —> seam.
• Disassembly:
• Dynamic instability.
• If GTP hydrolysis is faster than incorporation of new tubulin dimers, microtubules will
undergo catastrophe.
• GTP hydrolysis causes conformational change in microtubules.
• Compaction at longitudinal interface of tubulin dimers.
• Conformational change in α subunit.
• Resulting in strain on microtubule lattice —> disassembly.
• GDP-bound tubulin conformation forces the proto laments to peel and curve away.
• Bending causes the proto laments to break o .
• Newly formed microtubules undergo catastrophe less frequently.
• Not all GTP gets hydrolyzed —> microtubule can stop depolymerizing.
• Intrinsic polarity due to head-to-tail assembly:
• (+) end grows faster than (-) end.
• Directionality —> can be used as tracks.
• Molecular motors = kinesin & dynein.
• Repair:
• Self-repair if they lose tubulin dimers within the core.
• Self-repair if damaged by a laser.
• Microtubule organizing centre (MTOC):
• Centrosome.
• Microtubule (-) end anchored to the MTOC.
• Tubulin dimers are added to the (+) end.
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 • Microtubule-associated proteins:
Binds to
Type Example microtubule Functions:
…

• Stabilizes microtubules.
• Microtubules grow faster &
shorten slower when labeled with
Tau Length (side)
Tau —> catastrophe only 2% of
the time.
• Involved in Alzheimer’s disease.

• (+) end
• Seam in
yeast. • Can be phosphorylated @ Ser,
• Can also Thr, Tyr —> changes electrostatic
EB1-3 bind to the interaction at microtubule surface
side of • Regulates tubulin sheet closure.
microtubule • Strengthens the seam in yeast.
towards the
(+) end
Stabilizers • Loosely bound to Golgi
apparatus —> use CAMSAP to
capture free microtubules.
• Regulate (-) end dynamics.
• A ect non-centrosomal
microtubule organization &
numbers.
• CAMSAP1: tracks (-) ends.
(-) end of free
CAMSAP/Patronin/Nezah • CAMSAP2 & 3: deposited on (-)
microtubules.
ends, do not track.
• CAMSAP2: labels newly-
formed microtubule (-) ends.
• CAMSAP3: stabilizes broken
microtubule —> microtubules
do not depolymerize —> act
as seeds for new microtubule
growth.

• In round cells: found in MTOC

(centrioles).
• In polar cells: found in apex —>
centrioles are NOT MTOC in
Nucleators γ-tubulin ring complex (-) end
polar cells.
• Nucleates microtubules —> low
e ciency, other factors involved.
• Stabilizes microtubule (-) ends.
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 Binds to
Type Example microtubule Functions:
…

• Regulates the length of

CAMSAP2 at microtubules.
• Regulates (-) end growth.
• Recruited to microtubule by
ASPM.
• Cuts random lengths.
• Cut microtubules. Katanin • Can cut Taxol-stabilized
• Cause microtubule microtubules.
disassembly • 60-kDa subunit has enzymatic
• Members of AAA activity.
ATPase superfamily. • 80-kDa subunit binds
Severing • Localized to mitotic microtubules.
poles —> involved in • Upregulated by acetylation
mitosis.
• Cuts equal lengths along
• Monomeric in ADP.
microtubules.
• Oligomerizes in the
• Can cut Taxol-stabilized
presence of ATP. Spastin
microtubules
• Upregulated by polyglutamination
• Downregulated by detyrosination

• Cuts faster at (-) ends

Fidgeting • Can cut Taxol-stabilized
microtubules.
• Microtubule post-translational modi cations:
• Regulate motor movement & microtubule stability.
• Acetylation:
• Only on Lys —> most commonly on αK-40 which are on the inside of microtubules.
• Modi cation not accessible to MAPs or motors.
• Catalyzed by tubulin acetyltransferase aTAT1.
• Can get in from the free open ends of the microtubule.
• Can get in from breaks in the microtubule.
• Most likely binds to the microtubule surface, when a break occurs aTAT1 enters
the break and acetylates localized tubulin.
• Increases microtubule strength, stability, exibility, and resistance against mechanical
stress.
• Long-lived microtubules = more acetylation.
• Decreases spontaneous nucleation in vitro.
• Removed by deacetylases HDAC 6 & SIRT 2.
• HDAC 6 interacts with EB1 at (+) end.
• Detyrosination:
• Only on α-tubulin —> 1 Tyr residue @ C-terminal of most tubulin proteins.
• Increases microtubule stability.
• Catalyzed by decarboxypeptidase vasohibins (VASH) 1 & 2.
• Regulated by SVBP.
• Removed by tubulin tyrosine ligase (TTL).
• Rapidly re-tyrosinate all soluble tubulin dimers —> brings tubulin back to its
original state.
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 • Polyglutamination & polyglycylation:
• Can occur on both α & β tubulin.
• Addition of Glu/Gly to Glu residues towards the C-termini of tubulin.
• Cannot occur both at the same time.
• Increases microtubule stability.
• Microtubule drugs:
• Colchicine:
• From meadow sa ron.
• Binds to all tubulin dimers, prevents formation of new microtubules & re-elongation.
• Eventually results in no microtubule in the cell.
• Reversible e ects.
• Nocodazole:
• Synthetic.
• Binds to all tubulin dimers, prevents formation of new microtubules.
• Taxol:
• From Paci c Yew Tree.
• Binds and stabilizes microtubules, prevents depolymerization.
• Stops mitosis.
• Has been used for cancer treatment.
• Vinca alkaloids:
• From Madagascar Periwinkle plant.
• Bind to interface of tubulin heterodimers.
• Preferentially bind to unpolymerized β-tubulin —> block incorporation.
• Prevents microtubule assembly.
• Widely used for cancer treatments.
• Microtubule & actin:
• MAP-1:
• A subunit has 3 microtubule binding sites.
• B subunit has 1 microtubule binding site.
• Can also bind actin.
• Directly bind and cross-link microtubules & actin laments.
• WHAMM:
• WASP Homologue Associated with Actin, Membranes, and Microtubules.
• Located at membranes (e.g. Golgi).
• Associated with actin laments & microtubules.
• Promote actin lament nucleation by activating Arp2/3 complex.
• Pro lin:
• Co-distributes with microtubules.
• Increase lamellipodia formation = decreased microtubule-pro lin interactions.
• Formin:
• Link some pro lin to microtubules.
• Small amount of SMIFH2 or Dia2 siRNA causes a small but signi cant decrease in
pro lin-microtubule associations.
• Microtubules can be used for movement:
• Microtubules themselves can move intracellular material but only over short distances.
• Polymerizing microtubules can push.
• Depolymerizing microtubules can pull.
• Position nucleus in ssion yeast.
• Move chromosomes.
• Move ER.
• Usually happens on acetylated microtubules.
• ER docks on the microtubule and rides the polymerization towards the (+) end.
• Microtubule (+) end tracking protein EB1 can bind ER protein STIM 1.
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 Intermediate laments:
• Intermediate in diameter between actin laments & microtubules.
• Various proteins expressed by 70 di erent genes.
• Overexpressed in tumor cells.
• Expressed IF type is based on the di erentiated cells they arose from.
• Useful for diagnosing metastatic tumors.
• Functions:
• Provides mechanical support for multicellular animals.
• Attaching to 2 types of cell junctions:
• Desmosomes: transmits forces between cells.
• Hemidesmosomes: transmits forces between cells and ECM.
• Junction-intermediate lament network provides tissues with their mechanical integrity.
• Mutant mouse with truncated form of keratin-14 (found in basal layer of skin epidermis)
resulted in extensive blistering following mild trauma.
• Sequencing of keratin-14 in EBS patients showed a mutation in the conserved
Arg-125 residue.
• Structure:
• All IF proteins have α-helical coiled coil rod-like core.
• Variable N & C domains & amino acid sequences give IFs their unique features.
• 2 IF monomers form polar coiled-coil dimers.
• Antiparallel staggered tetramers form apolar proto laments.
• 2 proto laments form proto bril.
• 4 proto brils make a 10 nm IF.
• Apolar —> cannot act as tracks but can be transported as cargos.
• Assembly:
• Unknown mechanism in vivo.
• No known nucleating, capping, severing proteins.
• Very stable.
• Resist high temperatures, high [salt] and detergents.
• Phosphorylation:
• Major post-translational modi cation.
• Occur at many sites, usually during mitosis.
• Often destabilizing IF and block assembly.
• Phosphorylation of neuro laments (IF in neurons) stabilizes them.
• Keratin:
• α-keratin:
• Mainly α-helices.
• Hair, nails, horn, claws, hooves, mammalian skin, hag sh slime.
• β-keratin:
• Mainly β-sheets.
• Nails, features, beaks, reptile skin.
• IF-binding proteins — Plakins:
• Largest family of IF-binding proteins.
• All have plakin domains and/or plakin repeats.
• Eg. Plectin —> bind IF, actin laments, microtubules.
• Elastic:
• IF can stretch 33% of their size and return to native states.
• Can stretch to > 80% but α-helices turn into β-sheets —> do not recoil.
• A single IF can be stretched 250% before breaking.
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 • Cell mechanics:
• Vimentin-based IF stabilize the position of organelle and enhance elastic behaviour in
embryonic cells.
• Cytoplasmic IF associate with many organelles —> control their shapes & positions.
• Nucleus:
• Bind nuclear envelope through envelope proteins in the LINK complex.
• Bind through microtubule or actin-associated proteins that bind LINK.
• Outer nuclear membrane protein Nesprin-3 can bind IF through plectin.
• Mitochondria:
• Keratin, vimentin, desmin, neuro laments all bind mitochondria.
• In uence mitochondria distribution & metabolic functions.
• Golgi apparatus
• Cell motility:
• During actin-based motility:
• Keratins 6, 16, 17 are increased.
• Keratins 1, 10 are decreased.
• Invasive tumors have increase levels of IF proteins.
• Keratin 14 —> increased migration of breast cancer cells.
• Keratin 19 —> increased migration of hepatocellualr cancers but decreased migration
of breast cancer cells.
• IF depletion often causes a decrease in the speed of cell movement.
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