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Regulation of Nuclear Shape
Regulation of Nuclear Shape
A key current area of research in the laboratory is to identify molecular mechanisms regulating nuclear size and shape. We have carried out genetic and microscopy-based screens to identify components and pathways that regulate these processes.
In cells, the form and dimension of organelles are critical in defining their identity and function. The ratio of organelle size to cell size is maintained at a near-constant ratio. The morphology of budding yeast nuclei is kept close to a sphere during the interphase. However, it is also known to undergo remodelling during processes such as yeast growth, division and certain stresses. Studies show that protein and lipid distribution regulation at the nuclear envelope (NE) is crucial for preserving the nuclear shape and size. It also stresses how size and morphology are interlinked, altering one's influence on the other.
These studies have relevance in understanding how nuclear geometry is maintained under physiological conditions, how malleability in geometry required for development is achieved, and how abnormal morphologies arise in diseased conditions.
In a focused screen for components that regulate nuclear shape, in Saccharomyces cerevisiae we have identified several key processes that affect nuclear shape and NE organization. This includes the nucleolus, chromatin remodelers and components of the endoplasmic reticulum and vacuole (Male etal, 2020; Deolal et al, 2021, Deolal and Mishra, 2021). Altering nucleolar size triggered an asymmetric expansion of the nucleus, leading to blebs and extensions of the NE into the cytoplasm. We are now investigating how an increase in nucleolar size is sensed and communicated to promote NE biosynthesis and why nuclear expansion is isotropic in normal conditions but becomes non-isotropic in others. This is very important to know as gene expression can be affected by abnormal nuclear shapes, and also because chromosome organization is tightly coupled to association with the NE and INM proteins.
In all eukaryotes, chromatin organization within the 3-dimensional space of the nucleus is critical for all chromosome transactions like proper segregation of chromosomes to daughter cells, transcription, replication and repair of DNA. Nuclear extensions, blebs and other nuclear abnormalities are hallmarks of cancers, progeria, neurological defects, and multiple developmental disorders including muscular dystrophies.
It is still unknown as to how the NE expands uniformly during interphase in dividing cells and how this expansion is integrated with cell growth.Therefore, using the mutants and models we have, we seek to determine how nuclear expansion is coupled to cell growth and division, and what molecular pathways regulate the isotropic expansion of the nucleus.
Lipid metabolism and organelle morphology
Lipid metabolism and organelle morphology
Regulation of lipid biosynthesis is critical to maintaining NE shape and size. In our screen, we have identified candidates that regulate nuclear shape and function and also perturb lipid homeostasis. These players not only affect the nuclear shape, but some also affect nuclear division and morphologies and, potentially, functions of other organelles as well.
The NE is contiguous with the endoplasmic reticulum (ER) and can be considered a special (albeit compositionally distinct) region of the ER. Most steps of phospholipid biosynthesis take place predominantly in ER, with some specific steps that are restricted to mitochondria and Golgi. However, the NE has also been shown to be a site of phospholipid and storage lipid metabolism.
Phospholipid biosynthesis involves the flow of lipid and lipid intermediates between multiple organelles and regulating this process is critical for an adequate supply of phospholipids to all the membranes. Using some of the mutants identified in the screen, we seek to study how lipid flow between organelles is regulated and the consequences of perturbing these processes on cell physiology including cell division and cell survival in poor nutrient conditions.
Regulation of Nuclear Architecture
Regulation of Nuclear Architecture
We found that protein SUMOylation is crucial for gene silencing and the subnuclear localization of proteins. Chromatin is organized into euchromatin and heterochromatin, facilitating regulated protein expression. In Saccharomyces cerevisiae, heterochromatin-like loci exist at telomeres, HM loci, and rDNA loci, here transcriptional silencing makes nucleosomes inaccessible to the transcription machinery. Sir2, a conserved NAD+-dependent histone deacetylase, plays a vital role in silencing through different complexes, notably with Sir3 and Sir4 at the telomeres and HM loci, and in the RENT complex at rDNA.
Our genetic screen for understanding how these distinct nuclear compartments are established and maintained, identified SUMOylation as a key regulator affecting the mobility of Sir2. The E3 ligase Siz2 was found to modulate gene silencing in a locus-dependent manner (Nagesh et al.,2012). We discovered that Siz2 activity influenced Sir4 levels at telomeres, and while all Sir proteins were SUMOylated, the SUMOylation of Sir2 reduced its interaction with Sir4, leading to decreased telomeric silencing (Hannan et al, 2015). This also increased Sir2 accumulation at the nucleolus. In addition, we found distinct euchromatic targets for SUMOylated vs. unmodified Sir2 (Abraham, PhD thesis). Overexpression of the deSUMOylating enzyme Ulp1, led to a loss of telomeric silencing due to impaired recruitment of Sir3 to telomeres (Abraham and Mishra, 2018). This suggests that SUMOylation of different proteins in the heterochromatin has distinct outcomes. Moreover, perturbation of SUMO ligase levels affected rDNA stability, with SUMOylation modulating Tof2 and Fob1 levels, resulting in genomic instability at the rDNA locus (Abraham et. al, 2019).
Overall, these findings highlight the significance of protein SUMOylation in the compartmentalisation and stability of proteins within the nucleus of S. cerevisiae. The localisation of SUMOylation machinery i.e. the SUMO conjugating enzymes and the deSUMOylating enzymes to specific nuclear compartments, enables rapid responses to internal and external signals, influencing nuclear architecture.
To understand the dynamic regulation of nuclear architecture, we are now studying alterations to NE and nuclear compartments to the changes in the growth environment. Our genetic screens identified that perturbation of nutrient metabolic pathways alters nuclear shape and size (Chhaya Kispotta, unpublished observations). We are now investigating the specific signalling pathways involved in this and the consequences of altered nuclear organization on gene regulation.
Protein SUMOylation on Candida glabrata: a potential anti-fungal target
Protein SUMOylation on Candida glabrata: a potential anti-fungal target
Branching out from S. cerevisiae, we study SUMOylation in Candida glabrata, a yeast closely related to S. cerevisiae but an opportunistic pathogen with a high level of anti-fungal resistance. We have identified the protein SUMOylation and deSUMOylation machinery in C. glabrata (Gujjula et al, 2016 and Gupta et al., 2020). Loss of SUMO protein is lethal for C. glabrata and perturbation of the pathway reduces its capability to infect and survive in the host cells. Perturbation of protein deSUMOylation activates the proteostasis pathway and leads to heightened protein degradation, rendering the pathogen highly compromised for survival in host cells (Gupta et al., 2024). We are currently focusing on identifying the key targets of this proteolytic degradation and exploring the potential to target both the SUMO pathway and the downstream pathways for developing antifungal drug molecules.
Regulation of gene silencing and gene expression by transcription termination factors and consequences on cell-fate determination
Regulation of gene silencing and gene expression by transcription termination factors and consequences on cell-fate determination
While investigating the compartmentalisation of heterochromatin and telomeric silencing, we found that loss of rtt103, a transcription termination factor, leads to reduced gene silencing at the telomeres. Loss of rtt103 also made cells more vulnerable to DNA damage (Srividya et al, 2012). Loss of telomeric silencing was due to increased stability of sub-telomeric transcripts in the absence of Rtt103 and the two other members of the transcription termination complex, Rai1 and Rat1 (Kathirvel and Mishra, 2023). Without these factors, meiosis and sporulation are severely compromised while cells grow and divide mitotically.
This suggests that these transcription termination factors may play specific roles in executing the meiotic program and differentiation to spores. Our preliminary results indicate that these mutants are defective in establishing the meiotic program, and we are currently investigating the molecular mechanistic basis of this phenotype.
Pictorial Summary of Past Research
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