senior honors thesis

SPS1 and its function/pathway

What is the name of a gamete in budding yeast?

spores

What is the spindle (in meiosis)?

a micro tube-based structure used for chromosome segregation

What two distinct signal transduction pathways regulate spindle disassembly during meiosis II?

• Sps1 STE20-family GCKIII kinase

• Ama1, the meiosis specific activator of the APC/C

What is the APC/C?

(anaphase-promoting complex/cyclosome)

• large cellular machine that tags specific proteins for destruction so the cell can divide correctly

• triggers anaphase by destroying proteins that hold sister chromosomes together

What is Ase1 (PRC1 in mammals)?

an antiparallel microtubule bundling protein important for spindle formation and stabilization

What is the spindle midzone?

What is a STE20-family GCKIII kinase?

a member of a specific subgroup of serine/threonine protein kinases that help control cell growth, stress responses, and lipid metabolism

Sps1 is phosphorylated on ______, although the significance of that phosphorylation is not known

T174 —> threonine-174, a highly critical amino acid phosphorylation site on the Hog1 protein

What is Kip3?

(kinesin-8 protein) - acts as a microtubule depolymerase during spindle disassembly during mitosis

How did Aiden examine the importance of phosphorylation of T174 on Sps1? What did he discover?

By using CRISPR-based approaches to construct phosphorylation mutant strains that contained an SPS1 allele in which the threonine was changed to either alanine or aspartic acid. He discovered that in both mutants, spindle disassembly was disturbed

• but different phenotypes!

How does CRISPR-Cas9 work?

Like a DNA copy-and-paste; type of genome/gene editing

• Adapted from bacteria

• create a small piece of RNA with a short “guide” sequence that attaches (binds) to a specific target sequence in a cell’s DNA

  • also attaches to the Cas9 enzyme

• When introduced into cells, the guide RNA recognizes the intended DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location, mirroring the process in bacteria

• Once the DNA is cut, researchers use the cell’s own DNA repair machinery to add or delete pieces of genetic material, or make changes by replacing an existing segment with a customized DNA sequence

Why use Saccharomyces cerevisiae as a model organism?

Has a high level of conversation with humans and animals than other eukaryotic species

—> can be easily manipulated using genetic, genomic, and biochemical techniques

—> makes S. cerevisiae a powerful model organism to understand highly conserved basic cellular processes

How are diploid cells induced to undergo meiosis and sporulation?

When they are starved of adequate nitrogen and fermentable carbon sources

How many haploid spores are produced?

four

Where does sporulation occur?

Within the cytoplasm of the mother cell

What are prospore membranes (PSMs)?

phospholipid membranes that surround each haploid nuclei

What is the spindle pole body (SPB)?

A structure that duplicates at the beginning of meiosis I and meiosis II —> results in the formation of four SPBs

—> equivalent to the centrosome in humans

—> serves as the sole microtubule-organizing center for yeast

What is the spindle composed of?

Composed of kinetochore microtubules that attach to chromosomes and interpolar microtubules that interlock to provide stability and sliding function to the spindle

• microtubules emanating from the SPBs during meiosis I and meiosis II form the spindle

During meiosis II, the SPBs change composition and act as the initiation site for what structure? What happens after?

The initiation site of PSM formation

• after, the PSMs elongate from the SPBs to encapsulate the haploid nuclei —> then rounds up and closes up after spindle disassembly to become the plasma membrane of the spores

What is considered the cytokinetic event of meiosis in S. cerevisiae?

the closing of the PSMs

What does the cellularization of the spores require?

The coordination of the prospore membrane closure and other cellular events like spindle disassembly

When cells lack functional SPS1, what kind of defects are present?

defects in PSM development and spindle disassembly

• PSMs are hyper elongated, only produce 70% rounded and closed PSMs

• the spindle extends like wild type, but fails to fully dissemble at the end of meiosis II, leaving fragments persisting within the cell

Why were alanine and aspartic acid chosen for the mutant strains?

• alanine cannot be phosphorylated

• aspartic acid has a negative charge that can mimic the effect of phosphorylation

(Sps1) How did Aiden assess whether T174 was required for sporulation?

• placed both types of mutant cells in sporulation media for more than 24 hours

• result: neither produced spores

(Sps1) What was the difference in morphology between the alanine mutant and the aspartic acid mutant?

Both were hyper elongated, but:

• PSMs were less hyper elongated in the alanine mutant cells than in the aspartic acid

  • aspartic acid cells more similar in length to wildtype, but narrower

• Differences were seen qualitatively and will be interesting to quantify in future studies

(Sps1) What was qualitatively observed regarding spindle disassembly in the mutants?

• Spindles were hyperelongated in both phosphorylation mutants, spindles curving to follow the perimeter of the cell, unlike spindles found in sps1 cells

  • Spindle fragments also present

• In the alanine cells, the growth of the PSMs seemed unsynchronized with the elongation of the spindle

• Spindle length was not quantified, so it is unclear whether one mutant presents a different spindle disassembly defect from the other

During meiosis I and II, where does Cin8 localize to?

Cin8 localizes along the length of the meiotic spindle

• Cin8—> kinesin-5 family member, important for mitotic spindle assembly as well as microtubule sliding during anaphase spindle elongation

How is AMA1 related to Bim1?

• may be important in the degradation of Bim1 once it is removed from the spindles

• however, does not play a role in removing Bim1 from the spindles

Loss of SPS1 and AMA1 affect different targets. What are these targets?

• loss of SPS1 —> prolonged Bim1 localization at the spindle

• loss of AMA1 —> prolonged Ase1 and Cin8 localization at the spindle