Biology subjects

Iyer, J.

Publications and source records attributed to Iyer, J..

9 recordsLinked to original sources

bioRxiv2025

Dynamin regulates PLK-1 localization and spindle pole assembly during mitosis

Accurate mitosis is essential for genome stability. Although dynamin is best known for its role in vesicular trafficking, its functions during mitosis remain unclear. Here, we identify a mitotic role for the C. elegans dynamin homolog DYN-1 in regulating spindle assembly and the localization of the key mitotic regulators SPD-2 and PLK-1. Depletion of DYN-1 results in enlarged metaphase spindle poles and increased centrosomal SPD-2 and PLK-1 levels. These effects likely depend on DYN-1-mediated vesicular trafficking, as shown by assays with the dyn-1(ky51) allele. Additionally, a subset of DYN-1-depleted embryos exhibit altered PLK-1 localization at the midbody during late mitosis, correlating with midbody assembly defects. Together, these findings establish DYN-1 as a previously unrecognized regulator of spindle pole organization and early midbody assembly. SIGNIFICANCE STATEMENTDynamin is primarily known for its function in endocytosis, but its involvement in mitosis is not well defined. This study shows that dynamin helps organize mitotic spindle poles, ensures proper centrosome localization of SPD-2 and PLK-1, and supports early steps of midbody assembly in C. elegans embryos. These findings reveal a previously unrecognized mitotic role for dynamin and suggest new links between membrane dynamics, spindle organization, and the cytokinetic machinery.

cell biology↗

bioRxiv2022

A primary microcephaly-associated sas-6 mutation perturbs centrosome duplication and ciliogenesis in Caenorhabditis elegans

The human SASS6(I62T) missense mutation has been linked with the incidence of primary microcephaly in a Pakistani family, although the mechanisms by which this mutation causes disease remain unclear. The SASS6(I62T) mutation corresponds to SAS-6(L69T) in C. elegans. Given that SAS-6 is highly conserved, we modeled this mutation in C. elegans and examined sas-6(L69T) effect on centrosome duplication, ciliogenesis and dendrite morphogenesis. Our studies revealed that all the above processes are perturbed by the sas-6(L69T) mutation. Specifically, C. elegans carrying the sas-6(L69T) mutation exhibit an increased failure of centrosome duplication in a sensitized genetic background. Further, worms carrying this mutation also display shortened phasmid cilia, an abnormal phasmid cilia morphology, shorter phasmid dendrites and chemotaxis defects. Our data show that the centrosome duplication defects caused by this mutation are only uncovered in a sensitized genetic background, indicating that these defects are mild. However, the ciliogenesis and dendritic defects caused by this mutation are evident in an otherwise wild-type background, indicating that they are stronger defects. Thus, our studies shed light on the novel mechanisms by which the sas-6(L69T) mutation could contribute to the incidence of primary microcephaly in humans.

genetics↗

bioRxiv2021

The retromer complex regulates C. elegans development and mammalian ciliogenesis

The mammalian retromer is comprised of subunits VPS26, VPS29 and VPS35, and a more loosely-associated sorting nexin (SNX) heterodimer. Despite known roles for the retromer in multiple trafficking events in yeast and mammalian cells, its role in development is poorly understood, and its potential function in primary ciliogenesis remains unknown. Using CRISPR-Cas9 editing, we demonstrated that vps-26 homozygous knockout C. elegans have reduced brood sizes and impaired vulval development, as well as decreased body length which has been linked to defects in primary ciliogenesis. Since many endocytic proteins are implicated in the generation of primary cilia, we addressed whether the retromer regulates ciliogenesis in mammalian cells. We observed VPS35 localized to the primary cilium, and depletion of VPS26, VPS35 or SNX1/SNX5 led to decreased ciliogenesis. Retromer also coimmunoprecipitated with the capping protein, CP110, and was required for its removal from the mother centriole. Herein, we characterize new roles for the retromer in C. elegans development and in the regulation of ciliogenesis in mammalian cells, and suggest a novel role for the retromer in CP110 removal from the mother centriole.

cell biology↗

bioRxiv2021

The Chromatin Remodeling Protein CHD-1 and the EFL-1/DPL-1 Transcription Factor Cooperatively Down Regulate CDK-2 to Control SAS-6 Levels and Centriole Number

Centrioles are submicron-scale, barrel-shaped organelles typically found in pairs, and play important roles in ciliogenesis and bipolar spindle assembly. In general, successful execution of centriole-dependent processes is highly reliant on the ability of the cell to stringently control centriole number. This in turn is mainly achieved through the precise duplication of centrioles during each S phase. Aberrations in centriole duplication disrupt spindle assembly and cilia based signaling and have been linked to cancer, primary microcephaly and a variety of growth disorders. Studies aimed at understanding how centriole duplication is controlled have mainly focused on the post-translational regulation of two key components of this pathway: the master regulatory kinase ZYG-1/Plk4 and the scaffold component SAS-6. In contrast, how transcriptional control mechanisms might contribute to this process have not been well explored. Here we show that the chromatin remodeling protein CHD-1 contributes to the regulation of centriole duplication in the C. elegans embryo. Specifically, we find that loss of CHD-1 or inactivation of its ATPase activity can restore embryonic viability and centriole duplication to a strain expressing insufficient ZYG-1 activity. Interestingly, loss of CHD-1 is associated with increases in the levels of two ZYG-1-binding partners: SPD-2, the centriole receptor for ZYG-1 and SAS-6. Finally, we explore transcriptional regulatory networks governing centriole duplication and find that CHD-1 and a second transcription factor, EFL-1/DPL-1 cooperate to down regulate expression of CDK-2, which in turn promotes SAS-6 protein levels. Disruption of this regulatory network results in the overexpression of SAS-6 and the production of extra centrioles. Author SummaryCentrioles are cellular constituents that play an important role in cell reproduction, signaling and movement. To properly function, centrioles must be present in the cell at precise numbers. Errors in maintaining centriole number result in cell division defects and diseases such as cancer and microcephaly. How the cell maintains proper centriole copy number is not entirely understood. Here we show that two transcription factors, EFL-1/DPL-1 and CHD-1 cooperate to reduce expression of CDK-2, a master regulator of the cell cycle. We find that CDK-2 in turn promotes expression of SAS-6, a major building block of centrioles. When EFL-1/DPL-1 and CHD-1 are inhibited, CDK-2 is overexpressed. This leads to increased levels of SAS-6 and excess centrioles. Our work thus demonstrates a novel mechanism for controlling centriole number and is thus relevant to those human diseases caused by defects in centriole copy number control.

cell biology↗

bioRxiv2020

Functional assessment of the "two-hit" model for neurodevelopmental defects in Drosophila and X. laevis

We previously identified a deletion on chromosome 16p12.1 that is mostly inherited and associated with multiple neurodevelopmental outcomes, where severely affected probands carried an excess of rare pathogenic variants compared to mildly affected carrier parents. We hypothesized that the 16p12.1 deletion sensitizes the genome for disease, while "second-hits" in the genetic background modulate the phenotypic trajectory. To test this model, we examined how neurodevelopmental defects conferred by knockdown of individual 16p12.1 homologs are modulated by simultaneous knockdown of homologs of "second-hit" genes in Drosophila melanogaster and Xenopus laevis. We observed that knockdown of 16p12.1 homologs affect multiple phenotypic domains, leading to delayed developmental timing, seizure susceptibility, brain alterations, abnormal dendrite and axonal morphology, and cellular proliferation defects. Compared to genes within the 16p11.2 deletion, which has higher de novo occurrence, 16p12.1 homologs were less likely to interact with each other in Drosophila models or a human brain-specific interaction network, suggesting that interactions with "second-hit" genes may confer higher impact towards neurodevelopmental phenotypes. Assessment of 212 pairwise interactions in Drosophila between 16p12.1 homologs and 76 homologs of patient-specific "second-hit" genes (such as ARID1B and CACNA1A), genes within neurodevelopmental pathways (such as PTEN and UBE3A), and transcriptomic targets (such as DSCAM and TRRAP) identified genetic interactions in 63% of the tested pairs. In 11 out of 15 families, homologs of patient-specific "second-hits" enhanced or suppressed the phenotypic effects of one or many 16p12.1 homologs. In fact, homologs of SETD5 synergistically interacted with homologs of MOSMO in both Drosophila and X. laevis, leading to modified cellular and brain phenotypes, as well as axon outgrowth defects that were not observed with knockdown of either individual homolog. Our results suggest that several 16p12.1 genes sensitize the genome towards neurodevelopmental defects, and complex interactions with "second-hit" genes determine the ultimate phenotypic manifestation. Author SummaryCopy-number variants, or deletions and duplications in the genome, are associated with multiple neurodevelopmental disorders. The developmental delay-associated 16p12.1 deletion is mostly inherited, and severely affected children carry an excess of "second-hits" variants compared to mildly affected carrier parents, suggesting that additional variants modulate the clinical manifestation. We studied this "two-hit" model using Drosophila and Xenopus laevis, and systematically tested how homologs of "second-hit" genes modulate neurodevelopmental defects observed for 16p12.1 homologs. We observed that 16p12.1 homologs independently led to multiple neurodevelopmental features and weakly interacted with each other, suggesting that interactions with "second-hit" homologs potentially have a higher impact towards neurodevelopmental defects than interactions between 16p12.1 homologs. We tested 212 pairwise interactions of 16p12.1 homologs with "second-hit" homologs and genes within conserved neurodevelopmental pathways, and observed modulation of neurodevelopmental defects caused by 16p12.1 homologs in 11 out of 15 families, and 16/32 of these changes could be attributed to genetic interactions. Interestingly, we observed that SETD5 homologs interacted with homologs of MOSMO, which conferred additional neuronal phenotypes not observed with knockdown of individual homologs. We propose that the 16p12.1 deletion sensitizes the genome to multiple neurodevelopmental defects, and complex interactions with "second-hit" genes determine the clinical trajectory of the disorder.

genomics↗

bioRxiv2019

Drosophila models of pathogenic copy-number variant genes show global and non-neuronal defects during development

While rare pathogenic copy-number variants (CNVs) are associated with both neuronal and non-neuronal phenotypes, functional studies evaluating these regions have focused on the molecular basis of neuronal defects. We report a systematic functional analysis of non-neuronal defects for homologs of 59 genes within ten pathogenic CNVs and 20 neurodevelopmental genes in Drosophila melanogaster. Using wing-specific knockdown of 136 RNA interference lines, we identified qualitative and quantitative phenotypes in 72/79 homologs, including 21 lines with severe wing defects and six lines with lethality. In fact, we found that 10/31 homologs of CNV genes also showed complete or partial lethality at larval or pupal stages with ubiquitous knockdown. Comparisons between eye and wing-specific knockdown of 37/45 homologs showed both neuronal and non-neuronal defects, but with no correlation in the severity of defects. We further observed disruptions in cell proliferation and apoptosis in larval wing discs for 23/27 homologs, and altered Wnt, Hedgehog and Notch signaling for 9/14 homologs, including AATF/Aatf, PPP4C/Pp4-19C, and KIF11/Klp61F. These findings were further supported by tissue-specific differences in expression patterns of human CNV genes, as well as connectivity of CNV genes to signaling pathway genes in brain, heart and kidney-specific networks. Our findings suggest that multiple genes within each CNV differentially affect both global and tissue-specific developmental processes within conserved pathways, and that their roles are not restricted to neuronal functions. AUTHOR SUMMARYRare copy-number variants (CNVs), or large deletions and duplications in the genome, are associated with both neuronal and non-neuronal clinical features. Previous functional studies for these disorders have primarily focused on understanding the cellular mechanisms for neurological and behavioral phenotypes. To understand how genes within these CNVs contribute to developmental defects in non-neuronal tissues, we assessed 79 homologs of CNV and known neurodevelopmental genes in Drosophila models. We found that most homologs showed developmental defects when knocked down in the adult fly wing, ranging from mild size changes to severe wrinkled wings or lethality. Although a majority of homologs tested showed defects when knocked down specifically in wings and eyes, we found no correlation in the severity of the observed defects in these two tissues. A subset of the homologs showed disruptions in cellular processes in the developing fly wing, including alterations in cell proliferation, apoptosis, and cellular signaling pathways. Furthermore, human CNV genes also showed differences in gene expression patterns and interactions with signaling pathway genes across multiple human tissues. Our findings suggest that genes within CNV disorders affect global developmental processes in both neuronal and non-neuronal tissues.

genomics↗

bioRxiv2019

NCBP2-mediated apoptosis contributes to developmental defects of the schizophrenia-associated 3q29 deletion

The 1.6 Mbp deletion on chromosome 3q29 is associated with a range of neurodevelopmental disorders, including schizophrenia, autism, microcephaly, and intellectual disability. Despite its importance towards neurodevelopment, the role of individual genes, genetic interactions, and disrupted biological mechanisms underlying the deletion have not been thoroughly characterized. Here, we used quantitative methods to assay Drosophila melanogaster and Xenopus laevis models with tissue-specific individual and pairwise knockdown of 14 homologs of genes within the 3q29 region. We identified developmental, cellular, and neuronal phenotypes for multiple homologs of 3q29 genes, potentially due to altered apoptosis and cell cycle mechanisms during development. Using the fly eye, we screened for 314 pairwise knockdowns of homologs of 3q29 genes and identified 44 interactions between pairs of homologs and 34 interactions with other neurodevelopmental genes. Interestingly, NCBP2 homologs in Drosophila (Cbp20) and X. laevis (ncbp2) enhanced the phenotypes of homologs of the other 3q29 genes, leading to significant increases in apoptosis that disrupted cellular organization and brain morphology. These cellular and neuronal defects were rescued with overexpression of the apoptosis inhibitors Diap1 and xiap in both models, suggesting that apoptosis is one of several potential biological mechanisms disrupted by the deletion. NCBP2 was also highly connected to other 3q29 genes in a human brain-specific interaction network, providing support for the relevance of our results towards the human deletion. Overall, our study suggests that NCBP2-mediated genetic interactions within the 3q29 region disrupt apoptosis and cell cycle mechanisms during development. AUTHOR SUMMARYRare copy-number variants, or large deletions and duplications in the genome, are associated with a wide range of neurodevelopmental disorders. The 3q29 deletion confers an increased risk for schizophrenia, autism, and microcephaly. To understand the conserved biological mechanisms that are disrupted by this deletion, we systematically tested 14 individual homologs and 314 pairwise interactions of 3q29 genes for neuronal, cellular, and developmental phenotypes in Drosophila melanogaster and Xenopus laevis models. We found that multiple homologs of genes within the deletion region contribute towards developmental defects, such as larval lethality and disrupted cellular organization. Interestingly, we found that NCBP2 acts as a key modifier gene within the region, enhancing the developmental phenotypes of each of the homologs for other 3q29 genes and leading to disruptions in apoptosis and cell cycle pathways. Our results suggest that multiple genes within the 3q29 region interact with each other through shared mechanisms and jointly contribute to neurodevelopmental defects.

genomics↗

bioRxiv2017

Pervasive epistasis in cell proliferation pathways modulates neurodevelopmental defects of autism-associated 16p11.2 deletion

As opposed to syndromic CNVs caused by single genes, extensive phenotypic heterogeneity in variably-expressive CNVs complicates disease gene discovery and functional evaluation. Here, we propose a complex interaction model for pathogenicity of the autism-associated 16p11.2 deletion, where CNV genes interact with each other in conserved pathways to modulate expression of the phenotype. Using multiple quantitative methods in Drosophila RNAi lines, we identified a range of neurodevelopmental phenotypes for knockdown of individual 16p11.2 homologs in different tissues. We tested 565 pairwise knockdowns in the developing eye, and identified 24 interactions between pairs of 16p11.2 homologs and 46 interactions between 16p11.2 homologs and neurodevelopmental genes that suppressed or enhanced cell proliferation phenotypes compared to one-hit knockdowns. These interactions within cell proliferation pathways were also enriched in a human brain-specific network, providing translational relevance in humans. Our study indicates a role for genetic interactions within CNVs and identifies potential therapeutic targets for neurodevelopmental disorders.

genomics↗

bioRxiv2016

Protein phosphatase 1 down regulates ZYG-1 levels to limit centrioleduplication

In humans perturbations of centriole number are associated with tumorigenesis and microcephaly, therefore appropriate regulation of centriole duplication is critical. The C. elegans homolog of Plk4, ZYG-1, is required for centriole duplication, but our understanding of how ZYG-1 levels are regulated remains incomplete. We have identified the two PP1 orthologs, GSP-1 and GSP-2, and their regulators I-2szy-2 and SDS-22 as key regulators of ZYG-1 protein levels. We find that down-regulation of PP1 activity either directly, or by mutation of szy-2 or sds-22 can rescue the loss of centriole duplication associated with a zyg-1 hypomorphic allele. Suppression is achieved through an increase in ZYG-1 levels, and our data indicate that PP1 normally regulates ZYG-1 through a post-translational mechanism. While moderate inhibition of PP1 activity can restore centriole duplication to a zyg-1 mutant, strong inhibition of PP1 in a wild-type background leads to centriole amplification via the production of more than one daughter centriole. Our results thus define a new pathway that limits the number of daughter centrioles produced each cycle.\n\nAuthor SummaryThe centrosomes are responsible for organizing the mitotic spindle a microtubule-based structure that centers, then segregates, the chromosomes during cell division. When a cell divides it normally possesses two centrosomes, allowing it to build a bipolar spindle and accurately segregate the chromosomes to two daughter cells. Appropriate control of centrosome number is therefore crucial to maintaining genome stability. Centrosome number is largely controlled by their regulated duplication. In particular, the protein Plk4, which is essential for duplication, must be strictly limited as an overabundance leads to excess centrosome duplication. We have identified protein phosphatase 1 as a critical regulator of the C. elegans Plk4 homolog (known as ZYG-1). When protein phosphatase 1 is down-regulated, ZYG-1 levels increase leading to centrosome amplification. Thus our work identifies a novel mechanism that limits centrosome duplication.

cell biology↗