HT cell bio (development) Flashcards
Polarity and localisation
What are some main points to consider for polarity/asymmetry in cells (prokaryotes + eukaryotes), 9 main points
- Importance of polarity in both euks and proks (details in another card)
- polarity in bacteria and archea affecting cell division and protein distribution
- Identifying poles and midcells: the fundamental priciple involving FtsZ and the 3 models: DivIva, MinD, and GTPase switches
- Polarity in Eukaryotes:
- Yeast as model system (S.pombe and S.cervisiae)
- cdc42 mechanism, and cdc42 as a polarity module
- linking polarity to yeast development: bud formation, and Exogenous cues for schmoo formation and mating
- Linking asymmetry and polarity to cell fate through mating type switching and mRNA localisation
- the PAR (partition system) in C.elegans
what are the 3 examples for mechanisms of polarity in eukaryotes
- S.cerevisiae budding (yeast)
- C.elegans zygote
- Drosophila cell epithelium
what are 3 key points to remember about cdc42 (polarity module in eukaryotes) and positioning modules
- cdc42 is polarity module which is conserved across eukaryotes
- cdc42 is an organiser for polarity downstream, but it does not determine the position of polarity
- cdc42 is coupled to different positioning modules such as Bem1, PAR to determine position of polarity depending on cell type/species type
Linking polarity to budding and mating in yeast
what is yeast budding
- Yeast does cell division through budding (this applies for both sexual and asexual reproduction in yeast). In sexual reproduction yeast does an extra step of shmoo formation before budding. Budding occurs in all diploid and haploid yeasts and is the main reproductive strategy
Linking polarity to budding and mating in yeast
Describe the stages in bud formation (cell cycle) in yeast in terms of cdc42 localisation
Cell cycle and cdc42 localization
1. Early G1 Phase: in initial stage of growth phase. No bud present yet, cdc42 proteins also not present
2. late G1 phase: cdc42 proteins localise to a site on cell membrane. This localisation of cdc42 initiates polarity for bud formation
3. S/G2 phase: bud emergence starts. Cdc42 concentrated at site of emerging bud. DNA replication preparation (S phase) and enters G2 phase
4. G2/M phase: cell has distinct bud, cdc42 spreads around the bud/new cell and directs growth and division of cell from all sides.
5. Anaphase/telophase: cdc42 located at the new poles of cell, chromosomes segregate
6. telophase/Early G1 phase: cdc42 located at new pole of cells to finish division
Linking polarity to budding and mating in yeast
Describe the stages in bud formation (cell cycle) in yeast in terms of actin localisation
Actin Organization
1. Actin Patches (Arp2/3):
During Early G1 phase Actin patches contain the Arp2/3 complex is distributed in cell.
During Late G1 phase: it is concentrated at the emerging site of the bud (recruited by cdc42). It is involved in the initiation of actin filament branching and is found at the site of the emerging bud, it is organised/surrounds/recruited by Bni1 which is also at bud emergence site
- Actin Cables (Formins):
During S/g2 phase, Actin cables, which are long strands of actin filaments, are organized by formins (proteins like Bnr1 and Bni1). These cables guide vesicle transport to the growing bud site. Bnr1 is at site of cell division, and Bni1 is at the tipd of the emerging bud. During G2/M Bni spreads along the walls of new cell (same as cdc42 localisation), and Bnr1 is stil at cell division site - Actin Ring:
Eventually during anaphase telophase, an actin ring forms at the site of cytokinesis, which is where the cell will divide to separate the mother and daughter cells.
Linking polarity to budding and mating in yeast
Describe the stages in bud formation (cell cycle) in yeast in terms of septin ring localisation and dynamics
- Old Septin Ring:
Late G1: Septins are a group of proteins that form a ring at the future site of bud emergence and previous cytokinesis. The old septin ring is from the previous round of cell division. - New Septin Ring:
Early G2: A new septin ring forms at the site of the current bud emergence. Same site as cdc42 and bni1 - Septin Hourglass:
As the bud grows, the septin ring transitions into an hourglass shape. Localised at the new poles of the cells. Same as bnr1 and cdc42 - Split Septin Rings:
Finally, the septin structures splits into 2 septin rings, 1 ring in at the site of each new pole, marking the division between the mother and daughter cells. similar to the split of actin and cdc42 in last step
Linking polarity to budding and mating in yeast
Draw similarities between the location of cdc42, actin and septin in bud formation. Why might that be
cdc42 likely recruits actin and septin formation. cdc42 and bni1have same localisation from early G1 to G2/M by being at tip of bud emergence and then spreading to the entire new cell
cdc42, actin and septin are all located at site of division, hence are strongly involved in growth of the bud and division, through polarity
Linking polarity to budding and mating in yeast
Describe the mechanism and steps of axial budding and the proteins involved
- BUD3 and BUD4 are genes in yeast which allow cells to bud in a pattern called axial budding. Axial budding is when new buds form adjacent to the previous bud’s site
- Bud3p and Bud4p accumulate at the bud site as a double ring around the neck region, localized by the septins. Bud3p and Bud4p are proteins encoded by the BUD3 and BUD4 genes. They form a double ring structure around the division area of the mother and daughter cell, known as the neck.
- The septins, which are cytoskeletal proteins that form a ring at the bud site and interact w bud3 and 4, help position these proteins.
- Axl1/2 then associate at the neck and activate the Bud5 GEF:
Axl1 and Axl2 are proteins that interact with Bud3p and Bud4p. After this interaction, they activate Bud5, which is a guanine nucleotide exchange factor (GEF). GEFs are responsible for activating GTPases by accelerating GDP –> GTP in Cdc42 - Bud5 links the division landmarks (septin rings and bud3/4) to Cdc42 activation via a second GTPase cycle: Bud5 helps to transmit the positional information from the division landmarks (like the septin ring) to Cdc42, which is a small GTPase that regulates cell polarity. Cdc42 is activated at the correct location for the next budding event, ensuring that the bud forms in the proper position relative to the mother cell.
Linking polarity to budding and mating in yeast
What proof is there for BUD genes and axl1/axl2 and septin being involved in positional localisation and position for cell division in yeasts?
BUD mutants, where there is a mutation in the BUD genes. Yeast can still form normal buds, but they are placed incorrectly (not axial budding). This is depicted by the septins and the Axl2 proteins being mislocalized, which leads to the activation of Cdc42 at an incorrect site, resulting in a bud forming in the wrong position on the cell.
Linking polarity to budding and mating in yeast
why is it important to understand proteins involved in budding in yeasts in terms of polarity?
Understanding the process of bud formation and the role of these proteins is essential for insights into how cells control their shape and how they replicate in a structured manner. If cells don’t bud correctly, it could lead to problems with cell division and growth.
Linking polarity to budding and mating in yeast
Describe the molecular signalling pathway that regulated bud site selection and polarity organisation in yeast cells.
- landmark bud site selection module (positional module)
* division landmarks (which determine site of division) such as bud3/4, and septin landmarks activate Bud5 which is a GEF. Active Bud5 then activates Bud1/Rs1 by exchanging its bound GDP to GTP. It is thought that septin landmarks may activate cdc42
* Activated Bud1/Rsr1 then interacts w Bud2 ot can activate cdc24 and recruits cdc42 to division landmark. When it interacts w Bud2, a GAP, which converts bound GTP on Bud1/rsr1 back to bound GDP and inactivates bud1/rsr1.
2.cdc42 module (polarity module)
* when activated bud1/rsr1 interacts w calcium bound cdc24, and activates cdc24, which is a GEF for cdc42. It then activates cdc42 by GDP–>GTP. and cdc42 then directs septins, organization of actin cytoskeleton vesicle secretion to bud site for bud growth and establishment of polarity
* Cdc42 is then deactivated by binding to Bem3 which is a GAP and changes GTP to GDP
Therefore overall, positional landmarks like septin are though to signal and activate cdc42 for polarity and organizations of downstream effectors like actin and vesicles to help growth and division of buds
Linking polarity to budding and mating in yeast
Give an overall description of the FULL bud formation from positional landmark modules to polarity polarity modules (cdc42)
- bud3/4 determine the neck and position septins at division site. Septins become positional landmarks which determine and interact/activate polarity modules
- septin recruits and activates Bud5 GEF which activates Bud1/rsr1 and activates calcium bound cdc24
- activated cdc24 GEF activates Cdc42 which then acts as polarity module to activate and organise downstream proteins like actin and vesicles for budding and cell division
- cdc42 inactivated by Bem3 GAPs, and bud1/rsr1 inactivated by bud2 GAP
- all are activated and deactivated by GTP/GDP
Linking polarity to budding and mating in yeast
How can mating in yeast override usual landmark budding?
- In both S.pombe and S.cervisiae, during mating, shmoo formation shows and override of landmark polarity, because it is different to the landmarks usual landmarks in budding/division
- In S.pombe (fission yeast) shmoo formation The cells elongate towards each other, a process called in response to the presence of pheromones. This is a clear demonstration of changed polarity/override usual polarity as the cells need to orient themselves toward the source of the pheromones and away from their usual growth pattern.
- similarly in S.cerevisiae (budding yeast), They extend projections toward each other, which is a change in their normal polar growth pattern where they would typically form a bud.
- Overall: the normal landmarks for bud site selection is overriden by mating signal pathways. Mating pheromones cause cell to grow towards each other which overrides the power of the budding site landmarks. External signals like this can reorient cell polarity. Crucial for mating
Linking polarity to budding and mating in yeast
Describe the pheromone signalling in yeast s.cerevisiae
Pheromone signalling pathway is an MAPK pathway, and triggers shmoo formation.
During this pathway Ste2/Ste3 sensor activates Ste4/18 which activates Cdc42 and starts teh MAPK pathway leading to eventually the activation of Far1 protein.Far1 is responsible to stopping the cell cycle in G1 phase during mating, to allow mating to occur. This is done by Far1 binds to cyclin-Cdk complex and inhibits its activity, therefore stopping cell cycle for mating to occur. Far1 also is linked to cdc42’s activity and alters it during mating to allow shmoo formation instead of normal bud site formation/normal polarity site.
Linking polarity to budding and mating in yeast
Describe mechanism for how landmarks are over-ruled during shmoo formation
Similarly to the activation of polarity module cdc42. In this stage, during bud formation. In this mechanism for shmoo formation, mechanism is changed slightly. cdc24 is now influenced by location of pheromones and Far1, hence cdc42 will be recruited where Far1 and pheromones are detected for shmoo site selection modules, instead of responding to landmark bud selection modules and division landmarks like septin. The rest of the response and cdc42’s downstream impact stays same.
Overall: the cell normally uses these proteins to decide where to form a bud for asexual reproduction, but in the presence of mating pheromones, this system is repurposed. Instead of forming a bud, the cell forms a shmoo, which is a projection that grows toward the source of the pheromones for mating. Cells can redirect and reorganize normal growth processes in response to external signals, such as pheromones.
Linking asymmetry to cell fate
Describe the process during yeast mating, which causes the mother cell to switch mating types
- yeast cells have mating types. They can either be mating type ‘a’ or mating type ‘alpha’. 2 cells of OPPOSITE MATING TYPES NEED TO COME together to form a diplod zygote, or else no formation of zygote
- Before becoming a zygote, yeast cells either have ‘a’ or ‘alpha’, they then divide to become mother and daughter cells, which both posses ‘a’ mating type
- The mother cell then can switch its mating type from ‘a’ to alpha due to action of HO endonuclease.
- The mother cell ‘alpha’ then fuse w daughter cell ‘a’ to produce a diploid ‘alpha/a’ zygote.
- this allows more genetic diversity and allows mating to occur when there isn’t 2 types of mating types available to mate with.
Linking asymmetry to cell fate
What happens on a genetic level to allow mother cell mating type to switch
- only a part of the locus in the middle expresses th mating type, the 2 loci on 2 ends are silent.
1. HO endonuclease copies a HMLalpha silent loci into the expressed MAT locus and replaces the original HMRa
2. This allows the alpha mating type to be expressed as alpha instead of a in the mother cell.
Linking asymmetry to cell fate
Why is the HO endonuclease only active in mother cell?
- anaphase stage: both the mother and daughter cells have upstream regulatory sequences (URS1 and URS2) that are shown to inhibit HO endonuclease gene expression (HO off).
- telophase: a protein called Swi5 (Switching deficient 5) becomes active in the mother cell, and bingds to the URS1. However, the presence of Swi5 alone is not enough to activate the HO gene.
The daughter cell contains a protein called Ash1 (Asymmetric synthesis of HO), which inhibits the HO gene. This prevents the HO endonuclease from becoming active in the daughter cell, even in the presence of Swi5.
- late G1 phase: in the mother cell, another set of proteins, Swi4/6, joins Swi5 and binds to URS2. This combination activates the HO endonuclease gene (HO on). This allows for the mating type switching to occur.
Despite the presence of Swi5 in the daughter cell, the Ash1 protein continues to inhibit the HO gene, assisted by Swi4/6, maintaining the HO endonuclease as off.
Why is Ash1 protein only present in daughter cell
- ah1 proteinonly present in the daughter because ash1 mrna only present in the daughter cell due to asymmetry caused by cdc42 polarity
- ash1 mrna only present in daughter cell because the mrna forms a complex w a myosin motor protein (Myo4) encoded by SHE1 and adaptor proteins (which link myosin to mrna)
- due to the polarised actin cytoskeleton which is polarised by cdc42, the myo4 complex including the Ash1 mRNA is all localised into the daughter cell, giving it an asymmetric polarised distribution
How is the A/P axis in Drosophila ooyte established by localised mRNA determinantes?
future head (anterior) to the future tail (posterior) of an organism. In Drosophila, this axis is determined very early in the development of the oocyte (the cell that will become the egg).
Localized mRNA Determinants: Certain mRNAs are critical for the development of the A/P axis and are localized at specific ends of the oocyte:
- Bicoid mRNA (bcd-RNA): This is localized at the anterior pole of the oocyte and is involved in specifying the head and thorax of the fly.
- Oskar mRNA (osk-RNA): This mRNA is found at the posterior pole and is important for abdomen and germ cell formation.
- Transport Mechanisms: These mRNAs are transported to their respective poles within the oocyte via the microtubular cytoskeleton: Bicoid mRNA is transported by a protein called dynein, which typically moves toward the minus ends of microtubules, located at the anterior of the cell. Oskar mRNA is transported by kinesin, which moves toward the plus ends of microtubules, located at the posterior.
- Anchoring of mRNA: Once at their respective locations, these mRNAs are anchored in place by interactions with other proteins. This ensures that they remain localized where they can be translated into proteins that will dictate the developmental fate of the cells in those regions.
Describe to me the importance and the effect of the BICOID mRNA gradient/localisation on bicoid protein localisation/gradient and the hunchback protein gradient/localisation
- BICOID mRNA is localised at the anterioir, establishing initial concentration gradient
- Once fertilisation occurs, BICOID mRNA is then translated to BICOID protein which diffuses to other cells generating a concentration gradient from anterioir to prosterior. Vital for gene expression of other genes and developing the A/P axis
- BICOID protein binds to promoter of huncback gene and activates it. Its mRNA is distributed in a gradient with higher concentrations toward the anterior end. This is because the gradient of BICOID protein helps establish the gradient of HUNCHBACK mRNA by activating its transcription in the regions where BICOID concentration is high enough.
How is polarisation initiated in PAR system of C.elegans
- initiated by sperm entry site
- Sperm entry localizes PAR-2 to the posterior pole: When sperm enters the embryo, it triggers the localization of a protein called PAR-2 to the posterior side of the cell. This localization is crucial for establishing the A/P axis
- Actomyosin contractions generate a cortical flow and spread the PAR-2 domain: The actomyosin contractions are not just for muscle movement; in early embryos, they create a flow of cytoplasm that helps to spread proteins like PAR-2 across the cortex of the cell to reinforce polarity.
- PAR-2 and PAR-3 reciprocally inhibit each other’s localization: PAR-2 and PAR-3 are part of the partitioning defective (PAR) family of proteins. They set up opposing gradients in the cell, with PAR-2 in the posterior and PAR-3 in the anterior. They ensure that each stays in its respective domain by inhibiting the other’s localization to its domain.
- Polarity is maintained by a Cdc42–PAR-6–PKC-3 complex in the anterior pole: The proteins Cdc42, PAR-6, and PKC-3 form a complex that is maintained at the anterior side of the cell. This complex is essential for keeping the cell polarized after the initial polarity is established.
