D103 MT and Cytoskeleton (ALS 5, Videos 9 and 10)

in wt, the bacteria is dispersed, but without ActA, it is concentrated in one spot. which conclusion is best supported by the data?

ActA is necessary for the intracellular movement of listeria

• necessary bc it does not move at all without it; not sufficient

behavior of microtubules

dynamic

• MTs stem out the centrosome

• intrinsic property: dynamic instability

what is happening to MT A

it is undergoing catastrophy 

• growing and shrinking 

• depolymerization occurs bc no GTP cap (accumulation fo GTP-bound heterodimer) 

why do MTs not treadmill

because the MT (-) end is capped by the gamma-tubulin ring complex (gamma-TuRC) 

• - end is blocked by the complex; the complex forms the base for the MT to grow from 

• intrinsic: MT has dynamic instability with fixed - end 

mt growth in vitro is controlled by different MT binding proteins. which of the following statements is best supported by the data?

XMAP215 acts as a MT polymerase that accelerates + end growth

kinesin-13 acts as a depolymerase that leads to MT shortening at the + end

when both proteins are present, their activities partially oppose each other producing a small new growth

which way does dynein move

toward the - end (nucleus)

which way does kinesin move

moving toward + end (away from nucleus) 

how would you determine tha infection with Sars CoV-2 depends on transport along MTs

depolymerize MTs and look at the localization of the virus

lamellipodium

at leading edge of a migrating cell

• visualized by expression of gfp-tagged actin with FRAP

what do you conclude from a frap experiment 

the lamelipodium is highly dynamic actin network 

• frap assess for movement/ dynamic behavior 

• not told what is being added/ removed 

• need to depolymerize to repalce parts of MT, then repolymerize it

which reagents would be suitable to generate this image? kintochores are shown in red, dna in blue, and MTs in green. 

chicken anti-tubulin primary antibody, goat anti-chicken secondary with green fluorophore 

mouse anti-kinetochore primary, goat anti-mouse with red fluorophore 

MTs are made up of

tubulin heterodimers that consist of alpha and beta subunits

• the GTP found in the a-tubulin subunit = non-exchangeable (fixed)

• the gdp found in the B-subunit = exchangeable with soluble GTP

growth of a mt depends on

presence of a GTP cap

• the slight delay between addition of subunits and GTP hydrolysis on B-tubulin lead to the GTP cap

• GTP cap is NOT a capping protein for MTs (high enrichment of GTP subunits)

• 13 polar protofilaments form a MT

mts undergo 

dynamic instability 

• tubulin GDP or GTP only refers to B-tubulin 

• no building block = no cap = fraying = depolymerization

what controls mt dynamics

accessory proteins

gamma-TuRC

nucleates assembly and remains associated with the - end

• increases growth

stathmin 

binds subunits, prevents assembly 

kinesin 13

enhances catastrophic disassembly at + end

• destabilizes

katanin

severs microtubules

MAPs 

stabilize tubules by binding along sides 

XMAP215

stabilizes plus ends and accelerates assembly

+TIPs

remain associated with growing + ends and can link them to other structures, such as membranes 

where are MTs nucleated

centrosome (=MTOC)

• MTs radiate out

• centroles (2 or 4, depending on the stage of the cell cycle)

• pericentriolar matrix (PCM)

• other sites of MT nucleation: Golgi

pericentriolar matrix (PCM)

200 - 400 proteins

• ex: gamma-tubulin

• another MT at centrosome

• stabilizing = no depolymerization

(+) end binding protein

dynamically associates with the + end instead of the - end

kinesin-13

MT disassembling kinesin

stathmin 

MT destabilizing protein 

• promotes disassembly 

• nothing is added to (-) end because gamma-tubulin ring complex 

MT function

protein transport and organelle positioning

• an efficient long range transport system is required

• provide the tracks for motor-dependent transport to the axon terminal

kinesins

(+) end directed MT motor proteins

• protein family with functionally diverse members

• multimeric protein complex: 2 globular heads domains; ATP and MT binding; light chains → cargo binding

• most kinesins mediate movement toward + end

• requires ATP hydrolysis

dynein 

(-) end directed MT motor 

• very large protein 

• 2 heavy chain with globular head domains → ATP and MT binding 

• movement requires ATP hydrolysis 

• cargo recog depends on the dynactin adaptor (multimeric complex)

organelle positioning strictly depends on

functional MT motors

kinesin and ER membranes

kinesin moves ER membranes towards the plasma membrane

dynein and golgi membranes

dynein moves golgi membranes towards the cell center 

MT formation

• building block is a heterodimer composed of a and b-tubulin

• gtp on b0tubulin is hydrolyzed during filament assembly (GTP on a-tubulin is not hydrolyzed)

GTP cap

GTP hydrolysis on b-tubulin occurs with a slight delay after subunit addition

• with high conc of a/ b-tubilin heterodimer: GTP cap forms → MT growth

• lack of GTP cap (due to low a/ B-tubulin conc: MTs undergo depolymerization)

dynamic instability of MTs

intrinsic property of microtubules 

depend on the cytosolic conc of gtp-bound a/ b-tubulin heterodimer and the presence/ absence of the gtp cap 

mt nucleation at the centrosome

> 50% of MTs are nucleated at the centrosome

• their o ends are therefore capped and protected from depoly

• because of this - end capping mech, treadmilling i sless important for MTs

positioning of organelles

depends on MT motor proteins

• light chains or adaptors of these motor proteins are responsible for the selection of cargo (vesicles or organelles, such as the ER, golgi, or mito)

mt organization during interphase 

radial array of MTs, nucleated at the centrosome 

• organelle positioning 

• vesicle transport 

• do not differ from thise nucleated at golgi vs centrosome 

mt organization during mitosis

mitotic spindle, nucleated at the spindle poles (= centrosomes during mitosis)

• chromosome segregation

mts are less stable in mitosis than in interphase, but how is this regulated?

kinesin-13, XMAP215

• XMAP215 is phosphorylated in mitosis and p-XMAP215 cannot bind MTs

• using energy efficiently to find chromosomes

fluorescence recovery after photobleaching (FRAP) 

determine the dynamic behavior of a structure 

• expression of gfp in the cell → photobleach 

• measure how fast the gfp signal recovers at the site of photobleaching

interpretation of frap results

fast recovery: protein is in exchange with another pool of the same protein (cytosol)

slow/no recovery: protein does not exchange with another pool of proteins because it is relatively immobile

mitotic spindle

MTs form a complex structure during mitosis

• 3 different types of MTs in mitosis: astral MTs, kinetochore MTs, and interpolar MTs

characteristics of MTs in mitosis 

• decrease stability 

• enhanced nucleation 

• incraese in gamma tubulin in mito spindle 

astral MTs

position spindle pole at one side of the cell

kinetochore MTs

pulling chromosomes to different sides

interpolar MTs

push spindle poles apart 

taxol

chemotherapeutic that binds b-tubulin and prevents MT depolymerization

• stabilizes the MTs

• if they cannot depolymerize, tehy will grow and extend in one direction, then run out of building blocks → cell death

how do eukaryotic cells move

in a MT and dynein-dependent manner

• “crawling” (development, infection, repair) →actin-dependent mechanism

• swimming (sperm, protozoa, plasmodium)

flagellum of eukaryotic cells

MT-based structure 

• axoneme extends from mother centriole 

• 9 microtubule doublets that surround a central pair of singlet MTs (axoneme can be very long: 10-200 um) 

• MTs are cross-linked with each other; dynein 

motile cilia

cell surfance extensions related to flagella

• same 9+2 organization as eukaryotic flagella, but shorter 200/cell

• beat in synchrony to move fluid over surface (dynein-dependent = allow for movement)

• present in resp system, repro tract, and CNS

• disease: primary cilia dyskinesia (PCD) - impaired clearance of mucus and other particles

primary cilia

non-motile and function in signal transduction

• present on most non-dividing cells (one per cell)

• signaling organelle → contains many signaling molecules

• no central MT pair (no dynein → not motile)

• diseases: ciliopathies (polycystic kidney disease)

MTs during the cell cycle

dramatic reorg during cell division (centrosome-nucleated radial array in interphase vs mitotic spindle → blocking MT rearrangement is a powerful tool to block cell division → anti-cancer drug (taxol) 

FRAP

method to measure the dynamic behavior of a protein

• can be done in cells that express a gfp-tagged version of a protein of interest

• laser-mediate photobleaching destroys the fluorophore so that gfp no longer fluoresces, but the protein remains intact and functional

• measures how fast a protein is able to exchange with the same protein from a photobleached area

eukaryotic flagella overview

mediates movement of cells by a dynein and mt-dependent mech

• 9 mt doublets surrounding a central pair

• ciliary dynein (2 or 3 heavy chains)

• MTs are crosslinked so that atp-dependent dynein movement does not lead to MT sliding relative to one another, but to bending of the axoneme

cilia 

similar to flagella, but shorter, defects are linked to human disease (motile and primary cilia