Past Research

Graduate Research

The Bhalla lab is interested in how chromodealwithit2some structure and function contribute to chromosome segregation during cellular division. Using the nematode worm, C. elegans, we combine genetic and biochemical approaches with high-resolution microscopy and cytological techniques to gain a more informed view of how molecular events during meiosis and mitosis govern and are governed by higher-order chromosome behavior.

The ability to achieve successful cell division depends on accurate chromosome segregation to produce daughter cells with the correct chromosome complement. To ensure fidelity of this process checkpoint mechanisms coordinate events during cell division. Events that are monitored by a checkpoint must be completed in a given amount of time or the checkpoint will not be satisfied, resulting in cell cycle arrest or cell death [1]. Either of these responses reduces the probability that the aberrant cell division will produce daughter cells with the incorrect number of chromosomes. Defects in cell cycle checkpoints have been linked to tumorigenesis and cancer progression [2].

I specifically study how synapsis, a  process in meiotic prophase, is monitored and regulated to ensure proper segregation of homologous chromosomes.profil white

sexual reproduction

Meiosis is a specialized cell division in which cells undergo one round of DNA duplication and two successive rounds of division to produce haploid gametes. This process allows for the return to diploidy upon fertilization and is, therefore, required for sexual reproduction. Improper meiotic chromosome segregation can lead to an abnormal number of chromosomes in daughter cells. Zygotes that result from the fertilization of these aneuploid gametes are typically inviable. In some cases, the inheritance of an extra chromosome is not lethal but can result in serious developmental disorders such as, Down, Turner’s and Klinefelter’s syndromes or cancer predisposition [3].

In order to achieve proper meiotic chromosome segregation, homologous chromosomes must first pair and then synapse via the synaptonemal complex (SC), a proteinacious structure that assembles between each homologous pair. In the absence of synapsis, crossover recombination is either completely abrogated or severely reduced [4], resulting in missegregation of meiotic chromosomes and aneuploid gametes that contribute to infertility and birth defects.pairing synapsispairing

In C. elegans stably paired or synapsed chromosomes are monitored by a cell cycle checkpoint mechanism termed the synapsis checkpoint. This synapsis checkpoint requires active pairing centers (PCs), cis-acting sites near the end of each chromosome, that are also required for pairing and synapsis [5]. PCs promote pairing and synapsis by interacting with cytoplasmic microtubules and dynein through transient attachments to the nuclear envelope (NE), but how they activate the synapsis checkpoint is not currently understood.SAC

Like PCs, centromeres are specialized cis-acting chromosomal sites that nucleate transient structures to mediate microtubule binding, promote specific chromosome behavior and generate a checkpoint response. During mitosis, centromeres assemble kinetochores to orchestrate chromosome segregation [6]. Kinetochores are also required for the spindle assembly checkpoint (SAC), which monitors kinetochore-microtubule attachment and inhibits cell cycle progression until all chromosomes have successfully bioriented on the metaphase plate [7]. In addition, centromeres can act as sites for meiotic synapsis initiation in budding yeast [8, 9] and Drosophila [10, 11].

Because of the similarities between PCs and centromeres, I hypothesized that components of the SAC might act at PCs during meiotic prophase.

In so far I have shown that specific SAC proteins (MAD-1, MAD-2 and BUB-3) are required for the synapsis checkpoint and also negatively regulate synapsis in C. elegans. Mutation of SAC components suppress synapsis defects observed when dynein function is abrogated, potentially implicating SAC components in a tension-sensing mechanism that is thought to license synapsis during meiotic prophase. Our results suggest that the ability of some SAC components to monitor tension is conserved and may have been co-opted in a variety of biological contexts to promote genomic integrity.

Model for synapsis initiation in C. elegans. A pair of chromosomes (black lines) with PCs (red circles) interact with proteins at the nuclear envelope, including SUN-1 (blue), ZYG-12 (green) and dynein (purple), to gain access to the cytoplasmic microtubule network (orange lines). SAC components (yellow ovals) function at PCs. When a chromosome encounters another chromosome, homology is assessed by unknown mechanisms. If chromosomes are homologous and remain stably paired, they resist the pulling forces of the microtubule motor dynein, generating tension (black arrows between PCs) that is monitored by SAC components. Once sufficient tension has been generated (YES!), SAC components are removed, synapsis is initiated and the checkpoint is silenced. If chromosomes are not homologous, they cannot resist the pulling forces of dynein, are pulled apart and do not generate tension (NO!). Unsynapsed chromosomes then activate the synapsis checkpoint.

Model for synapsis initiation in C. elegans. A pair of chromosomes (black lines) with PCs (red circles) interact with proteins at the nuclear envelope, including SUN-1 (blue), ZYG-12 (green) and dynein (purple), to gain access to the cytoplasmic microtubule network (orange lines). SAC components (yellow ovals) function at PCs. When a chromosome encounters another chromosome, homology is assessed by unknown mechanisms. If chromosomes are homologous and remain stably paired, they resist the pulling forces of the microtubule motor dynein, generating tension (black arrows between PCs) that is monitored by SAC components. Once sufficient tension has been generated (YES!), SAC components are removed, synapsis is initiated and the checkpoint is silenced. If chromosomes are not homologous, they cannot resist the pulling forces of dynein, are pulled apart and do not generate tension (NO!). Unsynapsed chromosomes then activate the synapsis checkpoint.

My data along with other published data support a model in which these proteins inhibit synapsis initiation and promote checkpoint signaling by monitoring the stability of pairing, or tension, between homologous PCs. Previous work from the Bhalla lab suggests that stable PC pairing satisfies the synapsis checkpoint even when synapsis is prevented by mutation [12], lending support to this hypothesis that highly stable PC pairing is monitored by this meiotic checkpoint. Together these data support a model that tension generated by stable PC pairing is what is monitored by the synapses checkpoint. Once this pairing has generated sufficient tension to resist the pulling forces of the microtubule motor dynein, SAC proteins are either inactivated or removed from PCs to initiate synapsis and the checkpoint is silenced.

Given the remarkable similarities that exist between the synapsis checkpoint and the SAC, namely their use of cis-acting chromosomal loci to monitor chromosome behavior that depends on microtubule-mediated movement, further analysis of the role of these proteins may reveal conserved mechanisms that underlie the maintenance of genomic stability in both meiosis and mitosis.

References
1. Murray AW (1992) Creative blocks: cell-cycle checkpoints and feedback controls. Nature. 359: 599-604
2. Gollin SM (2005) Mechanisms leading to chromosomal instability. Semin Cancer Biol. 15: 33-42.
3. Hassold T and P Hunt (2001) To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet. 2: 280-291.
4. Bhalla N and AF Dernburg (2008) Prelude to a division. Annu Rev Cell Dev Biol. 24: 397-424.
5. MacQueen AJ, CM Phillips, N Bhalla, P Weiser, AM Villeneuve, et al. (2005) Chromosome Sites Play Dual Roles to Establish Homologous Synapsis during Meiosis in C. elegans. Cell. 123: 1037-1050.
6. Cheeseman, IM and A Desai (2008) Molecular architecture of the kinetochore-microtubule interface. Nat Rev Mol Cell Biol. 9:33-46.
7. Foley EA and TM Kapoor (2013) Microtubule attachment and spindle assembly checkpoint signalling at the kinetochore. Nat Rev Mol Cell Biol. 14:25-37.
8. Tsubouchi T, Macqueen AJ and GS Roeder (2008). Initiation of meiotic chromosome synapsis at centromeres in budding yeast. Genes & development. 22:3217-3226.
9. Tsubouchi T and GS Roeder (2005) A synaptonemal complex protein promotes homology-independent centromere coupling. Science. 308:870-873.
10. Takeo S, CM Lake, E Morais-de-Sa, CE Sunkel, and RS Hawley (2011) Synaptonemal complex-dependent centromeric clustering and the initiation of synapsis in Drosophila oocytes. Curr Biol. 21:1845-1851.
11. Tanneti NS, K Landy, EF Joyce and KS McKim (2011) A pathway for synapsis initiation during zygotene in Drosophila oocytes. Curr Biol. 21:1852-1857.
12. Deshong AJ, AL Ye, P Lamelza, and N Bhalla (2014) A quality control mechanism coordinates meiotic prophase events to promote crossover assurance. PLoS Genet. 10:e1004291.

cropped-web.jpg

Undergraduate Research

Regulation and function of Drosophila even-skipped
Adviser: Charles Sackerson
Department of Biology
California State University Channel Islands
Spring Semester, 2007

Expression of fushi-tarazu (dark gray bands) and even-skipped (brown bands) in a Drosophila Embryo.

Expression of fushi-tarazu (dark gray bands) and even-skipped (brown bands) in a Drosophila Embryo.

The aim of this project was to understand the mechanism by which segments form in Drosophila during development. I specifically studied even-skipped (eve), which is responsible for an early step in segment formation. Through antibody staining for Engrailed, we found that embryos lacking the Eve protein in stripe 1 also experienced a loss of their mandibular segment. Therefore, we showed that Eve in stripe 1 appears to be required for formation of the mandibular segment.

 

 

Sea mammal gene regulatory region sequencing
Adviser: Charles Sackerson
Department of Biology
California State University Channel Islands
Fall Semester, 2008

Blue Whale

Blue Whale

The aim of this project was to understand what causes these animals to have the ability to dive without oxygen for so long. The question we were asking was whether myoglobin expression increases as the animals mature in response to developmental changes, or whether the increase is induced by training, such as dive-induced hypoxia. We wanted to use PCR to attempt to isolate the regulatory region of myoglobin genes from several species of whales, dolphins, and porpoises. However, because these species had not been sequenced, I had to design primers using conserved bovine myoglobin regulatory regions as templates. The primers I designed lead to the successful isolation of regulatory regions of myoglobin genes in these sea mammals. This project was in collaboration with another group of students who were studying the behavioral aspect of this phenomenon. So far the project has been successful and is now aimed at investigating the differences in myoglobin between pigs and whales.

 

Investigating the use of conserved gene family, Sfrps, across embryos of vertebrate species
Science Foundation of Ireland Summer Undergraduate Research Fellowship
Advisor: Paula Murphy
Department of Zoology
Trinity College Dublin
Summer, 2009

Sfrp expression in chicken embryo

Sfrp expression in chicken embryo

 After completing my undergraduate degree, I accepted a summer research internship at Trinity College Dublin. During this time I worked in the Murphy lab exploring how differential use of Wnt modulators during development may have been involved in the diversification of the vertebrate body form. To do this I investigated the expression patterns of Wnt signaling modulators; secreted frizzled-related proteins (Sfrps) across embryos of two vertebrate species; mice and chicks. I performed detailed, systematic analysis of Sfrp1-4 expression throughout

Sfrp expression in mouse embryo

Sfrp expression in mouse embryo

various stages of development in the mouse and chick in four specific developing systems; the forelimb, hind limb, forebrain and hindbrain using whole mount in situ hybridization. Embryos were then reconstructed into 3D images using Optical Projection Tomography. I viewed the 3D gene expression data using Mouse Atlas Project software and collected sections of interest to analyze and compare. Comprehensive analysis revealed both similarities and differences in the spatial and temporal expression of these genes. The data I generated is concurrent with the hypothesis that differential use of Sfrps during development may have been involved in the diversification of the vertebrate body form.

 

Graduate Rotation Projects

Asymmetric Division in the Mammary Gland
Adviser: Lindsay Hinck
Department of Molecular, Cellular & Developmental Biology
University of California Santa Cruz
Summer, 2010

Dividing HeLa stained for DNA (blue) and NuMA (green)

Dividing HeLa cell stained for DNA (blue) and NuMA (green)

I specifically worked on finding ways to identify asymmetric divisions versus symmetric divisions in human breast cancer cells and mouse mammary tissue using specific asymmetric division markers. The hypothesis is that tumorigensis may be a result of excess symmetric divisions leading to increased proliferation. Therefore, cancerous tissues may have a higher percentage of symmetric divisions and a lower percentage of asymmetric divisions than healthy tissues. Unfortunately, I was never able to test this hypothesis because finding asymmetrically dividing cells proved difficult at first. However, the work I did on trouble shooting ways to identify asymmetrically dividing cells contributed to the ability of others in the lab to successfully visualize asymmetric divisions in both human and mouse breast tissues and they are now testing this hypothesis.

 

PCH-2 localization & SIR-2 checkpoint control in meiosis of C. elegans
Adviser: Needhi Bhalla
Department of Molecular, Cellular & Developmental Biology
University of California Santa Cruz
Fall Quarter, 2010

LEFT: C. elegans gonad stained for PCH-2 and axial chromosomal element HTP-3. RIGHT: Model for how PCH-2 activates the synapsis checkpoint.

LEFT: C. elegans gonad stained for PCH-2 and axial chromosomal element HTP-3. RIGHT: Model for how PCH-2 activates the synapsis checkpoint.

During my rotation in the Bhalla lab I was interested in identifying new components of the synapsis checkpoint in C. elegans. Initially I investigated  whether SIR-2 is required for the synapsis checkpoint. Sir2 has previously been shown to be required for Pch2 localization and meiotic checkpoint activation in yeast. Therefore, I analyzed whether SIR-2 is required for the synapsis checkpoint in C. elegans by scoring apoptosis in a synapsis checkpoint induced animal. I showed that a null mutation in sir-2 was unable to rescue the elevated levels of apoptosis seen when the synapsis checkpoint is induced. Therefore, SIR-2 is not required for the synapsis checkpoint in C. elegans. I attempted to analyze a role for PCH-2 localization in two other candidates shown to interact with PCH-2 in a yeast two hybrid analysis. My approach was to visualize PCH-2 localization using antibody staining after RNAi depletion of these candidates. Unfortunately, the RNAi treatment was never successful, therefore, results were inconclusive. The search for regulators of PCH-2 is still being pursued in the Bhalla lab.

 

Size Matters: A Model For Cell Growth Mediated Entry Into Mitosis
Adviser: Doug Kellogg
Department of Molecular, Cellular & Developmental Biology
University of California Santa Cruz
Winter Quarter, 2010

The next quarter I completed a rotation in the Kellogg lab investigating how cell growth and cell size are monitored and how they are involved in cell cycle events. I specifically studied the correlation between cell growth and phosphorylation of the mitotic regulator, protein kinase C (Pkc1) in budding yeast. The model is that GTPRho1 model 2ase, Rho1, travels on transport vesicles to the site of cell growth where it incorporates into the membrane and becomes active. This creates a signal proportional to growth where then active Rho1 induces phosphorylation of Pkc1, activating it and inducing mitosis. in order to test this model I first analyzed if Pkc1 phosphorylation corresponds with cell growth. To do this I looked at bud growth and phosphorylation of Pkc1 after release from G1. My data showed that Pkc1 undergoes phosphorylation that coincides with a loss of G1 cyclins, the emergence of mitotic cyclins and bud growth. Therefore, I showed that Pkc1 phosphorylation corresponds with cell growth. I then analyzed if Rho1 induces phosphorylation of Pkc1. I approached this question by visualizing Pkc1 phosphorylation after induction of a constitutively active Rho1. My data showed that Rho1 induces phosphorylation of Pkc1. Together, the data I produced supports the model that Rho1 produces a signal that is proportional to cell growth to induce mitosis.

Soma-to-germ transition and lifespan in C. elegans
Adviser: Susan Strome
Departmentof Molecular, Cellular & Developmental Biology
University of California Santa Cruz
Spring Quarter, 2011

Wildtype and synMuvB mutant C. elegans L1 staged larvae stained for P Granule proteins, PGL-1 & PGL-2. Arrow heads indicated P granules.

Wildtype and synMuvB mutant C. elegans L1 staged larvae stained for P Granule proteins, PGL-1 & PGL-3. Arrow heads indicated P granules. SynMuv B mutants show ectopic expression of P granules in their somatic tissues. Patrell, L., et al. (2011)

In the Strome Lab I investigated whether germ cell specific nuclear proteins, P granules, would extend life span if expressed in the somatic tissue in C. elegans. My hypothesis was that P granules add nuclear integrity to somatic cells when ectopically expressed leading to a longer life span. I first analyzed if long-lived mutants (e.g. daf-2) express P granule proteins in their somatic cells. I also analyzed if other mutants that express P granule proteins in their somatic cells are also long lived by performing  longevity assays using synMuv B mutants that ectopically express P granules. These questions are still under investigation in the Strome lab.