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2 - DC > 15-17: Woolner > Flashcards

15-17: Woolner Flashcards

1
Q

Describe how the spindles rotate during mitosis

A

The spindles rotate in metaphase, but stop during anaphase, locking in the direction of cell division

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2
Q

Describe symmetric and asymmetric cell division, and what they are used for

A

SYMMETRIC:
- Produces two daughter cells with the same size, components, and developmental fates
- Used to maintain and expand cell populations
- Can be used to spread/thin a tissue layer

ASYMMETRIC:
- Produces two daughter cells with different sizes OR components, and which follow different developmental fates
- Creates cellular diversity
- Can be used to thicken/stratify a layer
- Chromosomes are still divided equally

Note: Both these types can be seen in either polarised OR non-polarised cells

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3
Q

Give three examples of asymmetric division in animal physiology

A
  1. Mammalian skin - correct balance between symmetric and asymmetric in development
  2. C. elegans - first division of the embryo is asymmetric
  3. Drosophila - asymmetric divisions in neuroblasts (stem cells of nervous system)
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4
Q

What is Hertwig’s Rule?

A

That cells divide across their LONG axis (as spindles rotate to align with the long axis)

This remains true for most cell shapes, e.g., cone, triangle

If there is NO long axis (disc shape), then spindle orientation is random

There are a few exceptions (see other FC)

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5
Q

How can mitotic spindle orientation be observed and studied?

A

Cells can be grow on micro-patterned fibronectin structures, and spindle orientation can then be analysed by live cell imaging

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6
Q

Describe the important exceptions to Hertwig’s Rule

A
  1. Certain cell shapes (e.g., rectangle) don’t divide along their long axis - these exceptions actually proved helpful for understanding the process
  2. In some polarised cells, both symmetric and asymmetric divisions can occur against the long axis (e.g., Xenopus embryonic epithelial cells) - cell polarity adds an EXTRA level of control to division
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7
Q

Which two structures are key to aligning the spindle with the long axis (and how was this proven experimentally)?

A

Astral Microtubules (low doses of nocodazole - depolymerises astral microtubules only -> spindle now perpendicular instead of long axis)

Dynein (microinjection of function-blocking anti-dynein antibodies -> spindle fails to find long axis)

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8
Q

WHY does the spindle (almost always) align with the long axis in mitosis?

A

Dynein pulls on astral MTs (while still linked to the plasma membrane) to orient the spindle - WHERE the dynein is determines how the spindle is aligned

The pulling strength of dynein increases with Length of Astral Microtubules (a longer/denser MTs allow more dynein to be loaded on and pull the spindle)

Therefore, the greatest pulling strength is possible when the astral MTs are longest

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9
Q

How is Dynein linked to the Cell Cortex to orient the mitotic spindle?

A

Dynein (and its regulator dynactin) are recruited to the cell cortext by the NuMA-LGN-Gai complex

G-alpha-i: subunit of a heterotrimeric G-protein, bound to PM

LGN in vertebrates (or Pins in Drosophila, GPR1/2 in C. elegans) links Gai to NuMA

NuMA: Nuclear Mitotic Apparatus links LGN to Dynein-Dynactin (Mud in Drosophila, or LIN-5 in C. elegans)

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10
Q

In what other way do spindles consistently align, in addition to the long axis?

A

Parallel with the substratum (regardless of whether the cell is on a horizontal or vertical surface)

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11
Q

What are the consequences of overexpressing or mutating components of the NuMA-LGN-Gai complex?

A

Overexpression of ANY component -> increases spindle rotation, due to excess dynein (can show this by depleting actin, which reduces rotation back to normal levels)

Mutant Gai that can’t localise to PM -> spindle can no longer rotate or orient PARALLEL TO SUBSTRATE

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12
Q

How can polarised MDCK (Kidney) cells be grown?

A

Grow them in a gel, rather than on a coverslip
-> The cells will form polarised, spherical cysts instead of a monolayer, with the apical domain facing the cyst lumen, and the lateral domains facing the other cells in the cyst

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13
Q

When polarised MDCK cells are grown in a gel, how does the mitotic spindle align (and how is this affected by manipulating LGN)?

A

The spindles aligns parallel to the apical membrane, while LGN is enriched Laterally

If LGN is removed, the spindles align randomly

If LGN is artificially enriched on the apical membrane, spindle orientation is changed by 90 degrees

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14
Q

Why is LGN enriched only on the basolateral membrane in polarised MDCK cells?

A

Observations that need explaining:
- Par3 is needed to set up correct cell division axis
- Loss of Par3 causes LGN to be localised APICALLY as well as laterally
- BUT loss of Par3 does NOT affect Gai localisation - found on both membranes either way

Explained by cell polarity:
- Par3 and aPKC are localised apically, and aPKC phosphorylates any LGN that is found in the apical region
- Only unphosphorylated LGN in the lateral region can bind Gai
- Therefore, even though Gai is found everywhere, LGN can only bind to it at the basolateral membrane
- Therefore, NuMA -> Dynein -> Dynactin are ALSO only recruited laterally

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15
Q

Summarise some key BR points on the topic of Symmetrical Division

A

All from Di Pietro et al (2016):

  • Deregulation of spindle orientation affects fundamental developmental processes and homeostasis, and may be correlated with cancer
  • Three essential areas of regulation for spindle orientation - Cell Cortex, Recruitment of Force Generators, Astral MTs
  • Localisation of LGN complex is essential for MANY processes including in Drosophila neuroblasts, chick/mouse neuroepithelium, C. elegans zygotes and epithelial morphogenesis in mammals and flies
  • Some further proteins are thought to be involved in LGN localisation, though roles not fully understood yet (e.g., Afadin/Canoe, Disc Large 1, Huntingtin)
  • Diversity of recruiting mechanisms for LGN depending on the context (e.g., aPKC actually required for APICAL localisation of LGN in neuroblasts)
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16
Q

State why the first cell division in C. elegans embryos is asymmetrical

A

One spindle pole is displaced towards the posterior end of the cell, leading to a smaller P1 cell with all the P-granules, and a larger AB cell (the P-granules end up only in cells that give rise to eggs and sperm)

There is an unequal distribution of DETERMINANTS (e.g., proteins, RNA, P-granules) between the daughter cells, thus giving rise to progenitor cells for DIFFERENT TISSUES

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17
Q

Explain how the asymmetric first division of the C. elegans embryo is brought about

A

Observations:
- The spindle visibly oscillates (with the posterior pole being especially mobile)
- If you blast (OICD) the spindle pole, the fragments are pulled towards the cortex, and this occurs FASTER AT THE POSTERIOR [Grill et al, 2003]
- Embryos lacking LGN or Gai show very little movement of fragments, or oscillation of poles

  • Higher concentration of LGN found at posterior than anterior -> more pulling power on MTs
  • The Par proteins determine the localisation of LGN (in a similar mechanism to polarised epithelia) - LGN only binds at posterior where there is no Par3
  • The INITIAL POLARITY is set up by the entry of the sperm:
    -> The male pronucleus from the sperm provides an MTOC that nucleates MTs and begins to set up the embryo posterior
    -> MT growth causes Rho inactivation, and relaxation of actomyosin at posterior
    -> Retraction of actomyosin carries PAR proteins AWAY from the posterior
    -> THIS is what determines posterior localisation of LGN and asymmetric spindle alignment
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18
Q

Summarise how neurons arise from the Drosophila ectoderm

A
  • The ectoderm consists of epithelial cells that divide symmetrically (spindles align parallel with monolayer)
  • One neuroblast (nervous stem cell) arises within this tissue - this cell is selected by Notch/Delta signalling, and delaminates from the ectoderm
  • The neuroblast divides asymmetrically to produce one Ganglion Mother Cell and one more neuroblasts (differ in both size and determinants)
  • The Ganglion Mother Cell undergoes a TERMINAL division to generate two neurons (which won’t divide again)
  • Each neuroblast continues to undergo these asymmetric division (thus acting as stem cells)
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19
Q

Briefly describe the division of ectodermal epithelial cells in Drosophila

A

Symmetric division to maintain the ectoderm:
- Par3/aPKC/Par6 localised apically
- Fate determinants at basal end of cell
- (PREDICTED based on MCDK cells, but not actually proven yet) LGN, NuMA, Dynein/Dynactin localised laterally
- Spindle aligns parallel with ectoderm to give symmetric division

20
Q

Describe the division of the neuroblast in Drosophila

A

Although the neuroblast delaminates from the ectoderm, it maintains the same polarity (Par3/Par6/aPKC at apical, determinants at basal)

  • Initially, in early metaphase, the spindle forms parallel with the monolayer (as in ectoderm cell division)
  • However, during metaphase, the spindle rotates 90 degrees, to align with the cell polarity axis of the neuroblast and thus give an asymmetric division
  • This is due to APICAL localisation of LGN and NuMA in the neuroblast (counterintuitive, see other FC for explanation)
21
Q

WHY do LGN, NuMA and Gai localise in the same place as Par3/Par6/aPKC in the Neuroblast (whereas they don’t in the ectoderm or MDCKs)

A

In Neuroblasts, Inscuteable is expressed, which binds to Par3

Pins (Partner of Inscuteable, Drosophila homolog of LGN) binds to Inscuteable

This recruits the whole LGN complex to the apical surface

Dynein/Dynactin is recruited to the apical side, rotating the spindle

22
Q

Describe some of the fate determinants in Drosophila neuroblasts

A

1st Complex:
- Miranda (facilitates localisation of the rest of the complex)
- Prospero (TF that activates pro-neural genes in GMC)
- Brat (TSG that promotes cell cycle exit and differentiation)
- Staufen (binds prospero transcripts)

2nd Complex:
- Numb (Notch antagonist) and PON (Partner of Numb)

23
Q

Describe the type of divisions seen in mouse embryonic skin development

A

BOTH symmetric and asymmetric division occur:
- Asymmetric division produces a different apical cell that goes on to differentiate and generate stratified skin layers
- Symmetric division of basal layer expands and maintains the cell population as the embryo grows

24
Q

Describe the current model for the order of recruitment of components necessary for asymmetric division in mouse skin (and the supporting evidence for this model)

A

Gai (in PM) -> LGN/Pins -> NuMA -> Dynein/Dynactin

Knockdown of NuMA does prevent spindle alignment, but does NOT affect LGN Localisation

25
Q

Explain the importance of asymmetric cell division in mammalian skin, and how a knockdown can demonstrate this

A

Knockdown of LGN results in a thin, non-stratified, non-differentiated layer, as no asymmetric division can occur - thus asymmetric division is necessary for effective barrier function of the skin

In an LGN KD, blue dye can leak through the skin

26
Q

State the type of cell division seen in budding yeast

A

Asymmetric - mother and daughter cells are NOT identical

27
Q

Describe what is segregated asymmetrically in budding yeast, and explain why

A
  • Differentiation factors (e.g., Ash1 mRNA - mating-type-switching, isolated to daughter cell)
  • Aging determinants (daughter cells are born young, mother retains oxidatively damaged proteins etc)
28
Q

Describe how spindle positioning works in budding yeast

A
  • The spindle forms inside the nucleas, with MTOCs part of the nuclear membrane
  • As the bud grows, MTs extend into it
  • MT-pulling pulls the spindle and nucleus into the bud
  • Rather than Dynein, Myosin V interacts with astral MTs via Bim1 and Kar9, and walks along AFs to pull the microtubules
  • Once the MTs are in the bud, dynein (attached to PM via Num1) pulls on the MTs (via Pac1), and thus pulls one end of the nucleus into the bud
29
Q

Is the use of a Myosin in Spindle Positioning just a silly yeast thing?

A

Porbably not:

  • In addition to dynein, Myosin X may also play a role in mitotic spindle orientation in vertebrates
  • Myosin X can bind to actin via its head, and to MTs via its tail
  • These AFs are attached to the PM/cortex
  • Exact role is controversial and needs further study
30
Q

What determines where the cleavage furrow forms (and how was this shown experimentally)?

A

The position of the mitotic spindle determines the position of the cleavage furrow

Physical manipulations (i.e., pushing the spindle with a glass rod) caused the furrow to shift

31
Q

What generates the force for cytokinesis (and what determines where THIS happens)?

A

The Contractile Ring (composed primarily of F-actin and Myosin II) assembles just under the PM and generates the force for cleavage furrow formation

In Cytokinesis, arrays of F-actin and Myosin II constrict to generate the force that divides the cytoplasm

The MTs of the mitotic spindle specify the position of the actomyosin contractile ring (in addition to their role in separating the chromosomes) - this dual role ensures that these events are co-ordinated in space and time, and the ring will pinch the cell BETWEEN the separating chromosomes

32
Q

Briefly describe the three models for HOW the MTs of the mitotic spindle generate signals that influence the position of the contractile ring

A
  1. ASTRAL STIMULATION (Astral MTs carry furrow-inducing positive signals to the cell cortex)
  2. CENTRAL SPINDLE STIMULATION (the central spindle generates furrow-inducing positive signals) - the central spindle associates with many signalling proteins, including some that stimulate Rho
  3. ASTRAL RELAXATION (astral MTs promote relaxation of actomyosin at polar cortex, i.e. negative signal to prevent additional, polar furrows)
33
Q

Explain the experimental evidence for the 1st of the 3 models for how the MTs determine furrow position

A

ASTRAL STIMULATION - The Rappaport Furrow

  1. Embryo is manipulated by pushing a glass bead into it to create a “polo shaped” embryo
  2. Furrow forms on only one side of the cell, producing a binucleate egg
  3. As both nuclei enter mitosis, an additional furrow forms between the spindles on the other side of the cell, despite these spindles being merely adjacent, and not having a spindle equator

-> This is evidence of a positive signal from Astral Microtubules

34
Q

Explain the experimental evidence for the 2nd of the 3 models for how the MTs determine furrow position

A

CENTRAL SPINDLE STIMULATION:

A rat kidney epithelial cell was perforated on one side of the spindle, close to the spindle midzone

The furrow the ONLY formed on the UN-perforated side

This could be evidence of a signal from the central spindle - but unclear how such a signal would get to the cortex

35
Q

Explain the experimental evidence for the 3rd of the 3 models for how the MTs determine furrow position

A

ASTRAL RELAXATION:

Spindle Microtubules in C. elegans embryos were shortened (by stabilising Katanin, which severs MTs)

This resulted in ECTOPIC FURROWS - possibly because the negative (relaxing) signal could not be carried all the way to the polar cortex

36
Q

Which of the 3 models for how the MTs determine furrow position is likely to be correct?

A

It is probably a combination of all 3 models

The predominant model seems to vary by species and cell type

37
Q

What is the positive signal that specifies the contractile ring at the cell equator (and what is the experimental evidence for this)?

A

Rho GTPase (activator of myosin!)

  1. Zone of high Rho activity prior to furrowing in Xenopus embryo - zone remains tightly focused throughout cytokinesis [This was observed by injecting an embryo with a fluorescent probe for active, endogenous Rho]
  2. Moving the spindle using a needle shifts both the Rho activity zone AND the furrow
38
Q

Explain HOW Rho is activated at the cell equator

A

CENTRALSPINDLIN Complex:
- MKLP-1 (a plus-end-directed kinesin)
- MgcRacGAP

Centralspindlin accumulates at regions of MT overlap, and recruits Ect2 (a Rho GEF which activates Rho!)

MKLP1 is inhibited until anaphase by MCdk, preventing it from prematurely binding MTs

39
Q

What are the two locations where Centralspindlin accumulates?

A
  1. Overlapping MTs of the central spindle (shown by visualising Mgc in internal plane of cell)
  2. Equatorial Cell Cortex (Mgc accumulates at plus tips of MTs BEFORE cytokinesis is initiated)
40
Q

Summarise how the location of the actomyosin contractile ring in cytokinesis is specified

A

Centralspindlin and Ect2 accumulate at equator (due to overlapping spindle MTs)

This focuses the Rho activity zone at the equatorial cell cortex

Rho activates the actomyosin ring at the equatorial cell cortex

41
Q

If Mgc is an inhibitor of Rho, why is it found in a complex that defines the region of active Rho (include experimental evidnce)?

A

It was initially thought that the GAP activity of MgcRacGAP only functioned at the end of cytokinesis, to inactivate Rho and disassemble the contractile ring

HOWEVER, recent work has suggested that Mgc is required during cytokinesis to FOCUS the Rho activity zone

In experiments on early Xenopus embryos, Mgc KD led to broader, less focused or unstable Rho activity zones (depending on point mutation or deletion of GAP)

Like lots of things in biology, we need a negative signal as part of a cycle between positive and negative signalling

42
Q

Summarise the role of RhoA in regulating ring formation

A

After activation at the cell cortex by Ect2, and focusing by MgcracGAP:

  • RhoA activates FORMINS to nucleate long, unbranched AFs
  • RhoA promotes Myosin II activation and assembly by activating ROCK (Rho Kinase)
43
Q

Describe and explain the final stage of cytokinesis

A

ABCISSION - the splitting of one cell into two

  • The midzone of the spindle is remodelled to form the midbody (a densely packed region of MTs in the centre of the narrow bridge between daughter cells)
  • Contractile ring constricts until it reaches the MTs at the midbody
  • The midbody directs ABCISSION, which requires a membrane-associated ESCRT-III Filament System
  • The contractile ring cannot cut through the PM itself as it is beneath the PM - instead, to get through the densely packed midbody, it hands off to a membrane-associated ESCRT-III system, which narrows the connection between the cells (first on one side of the bridge, then the other)
  • Midbody is released into the cell (except in some cases where only one side is “snipped,” so one cell retains the midbody for signalling)
44
Q

Describe the roles of the ESCRT complexes in scission

A

First discovered in viral and vesicle budding

In cytokinesis, ESCRT-I is recruited first to the intercellular bridge to form membrane-associated rings on either side of the midbody ring

ESCRT-I then recruits ESCRT-III, which forms long filaments that coil around the site of membrane separation

ESCRT-III then narrows the connection between the daughter cells, eventually snipping one side of the bridge, then the other

45
Q
A

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