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Dynamic Cells > Dr. Woolner L15-L17 > Flashcards

Dr. Woolner L15-L17 Flashcards

1
Q

3 examples of asymmetric division

A
  1. Mammalian skin (uses symmetric too)
  2. First division of C. elegans embryo- nematode worm
  3. Drosophila neuroblasts- stem cells in fly nervous system
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2
Q

Hertwig’s rule

A

Cells divide across their long axis. Proposed by Hertwig in 1884. (division plane perpendicular to longest axis of the cell)

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3
Q

Polarised cell hertwig rule?

A

Symmetric and asymmetric divisions can occur along long axis instead of following hertwigs rule for example Xenopus embryonic epithelial cells.

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4
Q

Spindle contains..

A

3 groups of microtubules:

  1. Kinetochore MTs- connecting to chromatids
  2. Interpolar MTs- connecting to ones from other pole
  3. Astral MTs- from pole to edge of cell, very dynamic.

(these MTs emenate from the spindle pole, where the centrosome acts as an MTOC (MT organising centre)

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5
Q

Astral MTs

A

HIghly dynamic.

- end at spindle pole. + end out towards edge of cell towards cell cortex.

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6
Q

Dynein on astral MTs

A

Dynein moves towards minus ends so will move towards spindle pole. It’s found on the PM so it pulls the astral MTs towards the PM as it is fixed there. (generates cortical force on astral MTs)

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7
Q

Oconnel and wang 2000

A

Showed that astral MTs and dynein are needed to orient the spindle. Did this by adding low dose of Nocodazole-> only depolymerases the most dynamic MTs-> so Astral MTs.
The spindle could not move around and cell did not divide along long axis.

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8
Q

NuMA-LGN-Gαi complex

A

The NuMA-LGN-Gαi complex recruites dynein (and its regulator dynactin) to the cell cortex.
Galphai is subunit of G protein- anchors to the PM.
LGN links Galphai to NuMA. (Numa=pins in drosophila)
Numa links to Dynein which pulls the actin towards the PM.

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9
Q

Overexpress NuMA-LGN-Gαi complex?

A

Increases spindle rotation. This is due to excess dynein because (Kotak et al 2012) showed that depleting dynein activator, dynactin, reduces rotations back to control levels.

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10
Q

Gαi mutation

A

When Gαi can’t localise to the PM, spindle can no longer rotate or orient parallel to substrae. So if NuMA-LGN-Gαi can’t localise to the PM, spindle can’t orient properly.

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11
Q

MDCK cell

A

When grown in gel, from polarised spherical cysts, not monolayers. Spindles are aligned parallel to apical surfaces. LGN is enriched along lateral domains.
ZO-1= tight junction protein. Divides apical and lateral membranes.

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12
Q

Zo-1

A

ZO-1= tight junction protein. Divides apical and lateral membranes.

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13
Q

LGN mutation

A

Get randomly aligned spindles. Cant align properly.

If you artifically put lgn on apical membrane, spindles rotate 90 degrees.

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14
Q

Polar cells proteins found where?

A

Par3, par6, aPKC found on apical membrane. apkc phosphorylates and excludes lgl and par1.

Par 1, lgl, disc large and scribble found on basolateral membrane. Par1 phosphorylates and excludes par 3.

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15
Q

Knock out par3 in mdck cells?

A

Lose parallel alignment. Polarity lost and LGN normally on lateral. Now on apical and lateral.
But Galphai not affected, always on both apical and basolateral membranes.

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16
Q

LGN localisation

A

Determined by Par3 and aPKC.
Par3 is at apical with aPKC which phosphorylates LGN so it cant bind to G-ALPHAi
When Par3 mutated/ depleted- LGN binds to Galphai which is all over the pm so spindle moves randomly.

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17
Q

P granules

A

determinants- end up only in the cells that give rise to sperm and eggs.

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18
Q

C. elegans first division

A

Asymmetric in size and components.
One spindle pole is displaced towards posterior. After cytokinesis the P1 (posterior) cell is smaller than the AB cell. The p1 cell contains the P granules.
(Also the posterior pole of the spindle is much more dynamic, moves more.)

19
Q

OICD

A

OPtically-induced centrosome disintegration. Basically blasting the centrosome apart and watching how the fragments move.
Can see where most force generated in C elegans first division.

More force is generated at posterior cortex. The pole fragemnts move faster towards the posterior pole. Higher concentration of LGN at posterior pulling spindle. Dynein pulling it towards cortex more.

20
Q

Grill et al 2003- lgn and Galphai

A

Depleted LGN from embryos by RNAi. Blasted poles, no longer being pulled to cortex because lgn isnt holding dynein at cortex. same is true for embryos without galphai. Shows that these two are important for pulling spndle pole towards cortex.

21
Q

C elegans LGN localisation

A

The posterior of c elegans embryo is equivalent to the basolateral surface in polarised cells. there is more lgn in posterior where no Par3/par6/aPKC is present.
(apkc phosphorylates lgn so cant bind to galphai at anterior of embryo)

22
Q

Initial polarity in c elegans set up

A

Male pronucleus from sperm provides an MT organising centre (MTOC) that nucleates MTs and sets up posterior of embryo.
MT growth inactivate Rho and relaxes actomyosin at posterior. Retraction of actomyosin network carries the anterior par proteins away to the anterior. Sets up initial polarity in the on-cell embryo.

23
Q

Drosophila neuroblast divisions

A

Start with ectoderm of epithelial cells that divide symmetrically. Spindles align parallel with monolayer.

Neuroblast- divides asymmetrically to form; 1 neuroblast and 1 ganglion mother cell (GMC). Different size, different components and fate.

GMC: Undergoes terminal division to generate 2 neurones which won’t divide again.

Each neuroblast will continue to have these asymmetric divisions- making one self-renewing neuroblast and one GMC. So neuroblast=stem cell for fly nervous system.

24
Q

Neuroblast

A

Neuroblast is a stem cell

25
Q

Neuroblast cell division- asymmetricality..

A

IN epithelial cell0 par3/apkc/par6 at apical surface. Cell fate determinants localise to basal cell surface.

Neuroblast (selected by Notch/delta signalling pathway) delaminates from ectoderm, but retains polarity.

Spindle rotates to align with cell polarity axis in neuroblast. Only basal cell inherits cell determinants at bottom. One gets top one bottom.

26
Q

Why in drosophila neuroblast does spindle align with Par3/par6/apkc complex? giving asymmetric division

A

LGL (pins) and Numa to top of neuroblast.
They are in same place as par3/aplc/par6, whereas would expect them on basolateral side.

IN flies LGN=Pins= Partner of Inscuteable.
Neuroblasts express inscuteable which binds to par3/par6/apkc complex. Inscuteable then recruits LGN(pins/Galphai/Numa to apical surface. Means that more dynein/dynaactin is recruited to apical side of cell, rotating spindle to give asymmetric division.

27
Q

Numa and LGN in mammalian skin development

A

Need NuMA to localise LGn to give asymmetric division. In LGN knockdown or NuMA RNAi lose specialised differentiated skin- flattened skin. Not stratified.
Can see dye go through skin, not a good barrier.

28
Q

Budding yeat (sachromyces cerevisae cell division

A

Asymmetric because mother and daughter not identical.
ASH1 mrna segregated to daughter. Stops daughter switching mating-type.
Daughter doesn’t inherit ageing determinants from mother cell. (e.g. oxidatively damaged proteins).

29
Q

How does spindle get into bud in budding yeast?

A

Myosin V moves along actin filaments into bud, pulling astral MTs with it.
Remember myosin is an actin motor. not Mt.

Grabs the MTs by binding to the +TIPS proteins at the MT +end.
ONce MTs are in bud, dynein attached to the PM (via Num1) pulls on the MTs, pulling one end of the nucleus into the bud.

30
Q

Myosin in spindle positioning

A

Myosin V IN budding yeast. Also Myosin X in vertebrates. Binds to actin via its head and MTs via its tail. xact role of actin myosin in this is not certain, rquires further study

31
Q

cleavage furrow

A

Actin and myosin II form contractile ring to separate cells

32
Q

3 models for placing contractile ring?

A
  1. Astral stuimulation
  2. Central spindle stimulation
  3. Astral relaxation
33
Q

Ray rappaport

A

Performed experiments to investigate how the mitotic spindle determines position of cleavage furrow. Physical manipulations of the echinoderm embryo. Push spindle with glass rod, changes where furrow forms.

34
Q

Contractile ring

A

Just under PM, attached to PM.
Composed of primarily F-actin and Myosin II.

When cells enter mitosis, the interphase arrays of F-actin and myosin II dissassemble. So contractile ring is main array of F actin and mysoin and separates the two cells.

35
Q

Astral stimulation evidence

A

The Rappaport furrow.
Use sand dollar embryos, Took one cell embryo and put glass bead in the middle which displaced spindles. Furrow then formed only on one side of egg. Both nuclei enter mitosis. Then get 3 cleavages, suggesting spindles are sending positive cue to positiion furrow next to them.

36
Q

central spindle stimulation evidence

A

Rat kidney epithelial cell. Made hole in one side. The furrow forms fine on one side of cell but not on side with hole. So signal from centre of spindle can;t get there when there’s a hole in cytoplasm.

37
Q

Astral relaxation evidence

A

C. elegans embryo- spindle mts were shortened by stabilising katanin (which severs MTs). This resulted in ectopic furrows- suggested that astral MTs might provide a cur to relac the cortex. when they cant reach as far the cortex, more furrows are formed. contradicts first model.

38
Q

Rho gtpase in cleavage furrow

A
  1. Activates myosin 2
  2. Activates formin mDia- which will nucleate filaments of actin and the myosin 2 will form this contractile network.

Active Rho zones at equator region which activate these.

39
Q

Rho zones

A

Active rho zones seen just before cleavage furrow. if spindles moved, the active rho zone and cleavage furrow move too.

40
Q

Centralspindlin

A

A complex of MgcRacGAP and MKLP-1 (a kinesin-6) accumulates at regions of MT overlap.

Centralspindlin recruits the Rho GEF Ect2 to the central spindle.

(plus end kinesin so moves away from centre of spindle towards middle of cell or out towards cortex)

Imagine with Mgb-GFP can see green of MGcRacGAP at equator region where there’s conc of plus end MTs.

Ect2 and centralspindlin activate Rho to specify position of contractile ring. Ect2’gef activitiy is concentrated here.

41
Q

MgcRacGAP focussing Rho zone

A

The GAP activity inactivates Rho. So that Rho only active at this furrow where it’s needed.

Mutation in the GAP domain of MgcRacGAP show less focussed Rho activity zone. Don’t form furrow properly.

42
Q

regulation of contractile ring by RhoA

A

Rho activated by Ect2. Rho-GTP promotes F-actin assembly by activating formins- nucleate long/unbranched actin filaments.
Rho-GTP promotes myosin II activation and assembly by activating ROCK which phosphoylates myosin II

43
Q

Abscission

A

Final stage when cell splits into 2 daughters.
Central region of spindle remodelled into midbody- dende MTs and proteins in narrow intracellular bridge between daughters.
Midbody directs abscission, final membrane separation process, which requires membrane=associated ESCRT-III filament system.

44
Q

ESCRT-III filament system

A

ESCRTI and III
recruited to intracellular brindge to form membrane-associated rings on either side of midbosy.
escrtI recruits escrt III
escrt III forms long filament and wraps around site of separation and snips on both sides. Physically separates cells!

Dynamic Cells (5 decks)

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