Dr. Woolner L15-L17 Flashcards
3 examples of asymmetric division
- Mammalian skin (uses symmetric too)
- First division of C. elegans embryo- nematode worm
- Drosophila neuroblasts- stem cells in fly nervous system
Hertwig’s rule
Cells divide across their long axis. Proposed by Hertwig in 1884. (division plane perpendicular to longest axis of the cell)
Polarised cell hertwig rule?
Symmetric and asymmetric divisions can occur along long axis instead of following hertwigs rule for example Xenopus embryonic epithelial cells.
Spindle contains..
3 groups of microtubules:
- Kinetochore MTs- connecting to chromatids
- Interpolar MTs- connecting to ones from other pole
- 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)
Astral MTs
HIghly dynamic.
- end at spindle pole. + end out towards edge of cell towards cell cortex.
Dynein on astral MTs
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)
Oconnel and wang 2000
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.
NuMA-LGN-Gαi complex
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.
Overexpress NuMA-LGN-Gαi complex?
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.
Gαi mutation
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.
MDCK cell
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.
Zo-1
ZO-1= tight junction protein. Divides apical and lateral membranes.
LGN mutation
Get randomly aligned spindles. Cant align properly.
If you artifically put lgn on apical membrane, spindles rotate 90 degrees.
Polar cells proteins found where?
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.
Knock out par3 in mdck cells?
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.
LGN localisation
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.
P granules
determinants- end up only in the cells that give rise to sperm and eggs.
C. elegans first division
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.)
OICD
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.
Grill et al 2003- lgn and Galphai
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.
C elegans LGN localisation
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)
Initial polarity in c elegans set up
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.
Drosophila neuroblast divisions
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.
Neuroblast
Neuroblast is a stem cell
