Biology subjects

Krishnan, H.

Publications and source records attributed to Krishnan, H..

7 recordsLinked to original sources

bioRxiv2024

The conserved biochemical activity and function of an early metazoan phosphatidylinositol 5 phosphate 4-kinase regulates growth and development.

The ability to co-ordinate function between multiple cells is a critical requirement for multi-cellularity. This co-ordination is mediated by hormones or growth factors, molecules secreted by one cell type that can convey information to the other cells and influence their behaviour. Hormone-dependent signalling is mediated by second messenger systems;phosphoinositides (PIs) generated by lipid kinase activity are one such key second messenger system. Phosphatidylinositol 5 phosphate 4-kinase (PIP4K) is a lipid kinase that phosphorylates phosphatidylinositol 5-phosphate (PI5P) to generate phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2]. Following a comprehensive bioinformatics analysis of ca. 23296 proteomes covering the tree of life, we find that PIP4K is a metazoan-specific enzyme, although its homologs are also found in choanoflagellate genomes. To understand their function in early metazoans, we experimentally analysed the biochemical activity and physiological function of PIP4K from several early metazoans. We find that the PIP4K enzyme from an early branching metazoan sponge Amphimedon queenslandica (AqPIP4K), regarded as the earliest evolved metazoan, shows a biochemical activity highly conserved with human PIP4K; AqPIP4K is able to selectively phosphorylate PI5P to generate PI(4,5)P2 just as effectively as the human enzyme. Further, AqPIP4K was able to rescue the reduced cell size, growth and development phenotype in larvae of a null mutant in Drosophila PIP4K. These phenotypes are regulated through activity of the hormone insulin, acting via the cell surface insulin receptor, a member of the receptor tyrosine kinase family, that is unique to metazoans. Together, our findings indicate that in early metazoans, AqPIP4K is likely to function in a signal transduction pathway that is required for receptor tyrosine kinase signalling. Overall, our work defines PIP4K as a signal transduction motif required to regulate receptor tyrosine kinase signalling for intercellular communication in the earliest forms of metazoa.

biochemistry↗

bioRxiv2024

Ca2+ binding to Esyt is required to modulate membrane contact site density in Drosophila photoreceptors

Membrane Contact Sites (MCS) between the plasma membrane (PM) and endoplasmic reticulum (ER) have been shown to regulate Ca2+ influx into animal cells. However, the mechanisms by which cells modulate ER-PM MCS density is not understood and the role of Ca2+, if any, in regulating this process is not known. We report that in Drosophila photoreceptors, MCS density is dependent on the activity of the Ca2+ permeable channels-TRP and TRPL. This regulation of MCS density by Ca2+ is mediated by extended synaptotagmin (dEsyt), a protein localised to ER-PM MCS in photoreceptors and previously shown to regulate MCS density. We find that the Ca2+ binding activity of dEsyt is required for its functional activity in vivo. dEsytCaBM, a Ca2+ non-binding mutant of dEsyt is unable to modulate MCS structure in a manner equivalent to its wild type counterpart. Further, when reconstituted in dEsyt null photoreceptors, in contrast to wild type dEsyt, dEsytCaBM is unable to rescue ER-PM MCS density and other key phenotypes. Finally, when expressed in wild type photoreceptors, dEsytCaBM phenocopies loss of dEsyt function. Taken together, our data supports a role for the Ca2+ binding activity of dEsyt in regulating the ER-PM MCS density in photoreceptors thus tuning signal transduction in response to Ca2+ influx triggered by ambient illumination. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/588361v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@f8faacorg.highwire.dtl.DTLVardef@114009org.highwire.dtl.DTLVardef@8337f0org.highwire.dtl.DTLVardef@cd7c2f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

IMPA1 dependent regulation of plasma membrane phosphatidylinositol 4,5-bisphosphate turnover and calcium signalling by lithium

Lithium (Li) is a widely used as a mood stabilizer in the clinical management of Bipolar Affective Disorder (BPAD). However, the molecular targets of Li in neural cells that underpin its therapeutic effect remain unresolved. Inositol monophosphatase (IMPA1), is an enzyme involved in the resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) following receptor-activated phospholipase C (PLC) signalling. In vitro, Li inhibits IMPA1, but the relevance of this inhibition within neural cells remains unknown. Here we report that in human cells, treatment with therapeutically relevant concentrations of Li reduces receptor activated calcium release from intracellular stores and delays the resynthesis of PIP2 following receptor activated PLC signalling. Both these effects of Li are abrogated in cells where IMPA1 has been deleted. We also observed that in human forebrain cortical neurons, treatment with Li results in reduced neuronal excitability as well as reduced calcium signals following receptor activated PLC signalling. Following Li treatment of human forebrain cortical neurons, transcriptome analyses reveal downregulation of multiple components of the glutamate receptor signalling system. Glutamate is a key excitatory neurotransmitter in the human brain and thus our findings provide an insight into the mechanisms underlying the dampening of neuronal excitability following Li treatment. Collectively, our findings suggest that Li inhibits receptor activated PLC signalling leading to an altered transcriptional response and reduced neuronal excitability.

neuroscience↗

bioRxiv2022

PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

Phosphatidylinositol 3-phosphate (PI3P) and Phosphatidylinositol 5-phosphate (PI5P) are low abundant phosphoinositides crucial for key cellular events such as endosomal trafficking and autophagy. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) is an enzyme that regulates PI5P in vivo but can act on both PI5P and PI3P, in vitro. In this study, we report a novel role for PIP4K in regulating PI3P levels in Drosophila tissues. Loss-of-function mutants of the only PIP4K gene in Drosophila (dPIP4K29) show reduced cell size in larval salivary glands. We find that PI3P levels are elevated in dPIP4K29 tissues and that reverting PI3P levels back towards wild type, without changes in PI5P levels, can also rescue the reduced cell size phenotype. dPIP4K29 mutants also show an upregulation in autophagy and the reduced cell size can be reverted by decreasing Atg8a, that is required for autophagosome maturation. Lastly, increasing PI3P levels in wild type salivary glands can phenocopy the reduction in cell size and associated upregulation of autophagy seen in dPIP4K29. Thus, our study reports for the first time, a role for a PIP4K-regulated PI3P pool in the control of autophagy and cell size regulation that may explain the reported role of PIP4K in regulating neurodegeneration and tumour growth.

cell biology↗

bioRxiv2021

Genomic sequencing of Lowe syndrome trios reveal a mechanism for the heterogeneity of neurodevelopmental phenotypes

Lowe syndrome is an X-linked recessive monogenic disorder resulting from mutations in the OCRL gene that encodes a phosphatidylinositol 4,5 bisphosphate 5-phosphatase. The disease affects three organs-the kidney, brain and eye and clinically manifests as proximal renal tubule dysfunction, neurodevelopmental delay and congenital cataract. Although Lowe syndrome is a monogenic disorder, there is considerable heterogeneity in clinical presentation; some individuals show primarily renal symptoms with minimal neurodevelopmental impact whereas others show neurodevelopmental defect with minimal renal symptoms. However, the molecular and cellular mechanisms underlying this clinical heterogeneity remain unknown. Here we analyze a Lowe syndrome family in whom affected members show clinical heterogeneity with respect to the neurodevelopmental phenotype despite carrying an identical mutation in the OCRL gene. Genome sequencing and variant analysis in this family identified a large number of damaging variants in each patient. Using novel analytical pipelines and segregation analysis we prioritize variants uniquely present in the patient with the severe neurodevelopmental phenotype compared to those with milder clinical features. The identity of genes carrying such variants underscore the role of additional gene products enriched in the brain or highly expressed during brain development that may be determinants of the neurodevelopmental phenotype in Lowe syndrome. We also identify a heterozygous variant in CEP290, previously implicated in ciliopathies that underscores the potential role of OCRL in regulating ciliary function that may impact brain development. More generally, our findings demonstrate analytic approaches to identify high-confidence genetic variants that could underpin the phenotypic heterogeneity observed in monogenic disorders.

genomics↗

bioRxiv2020

Interdomain interactions regulate the localization of a lipid transfer protein at ER-PM contact sites

During phospholipase C-{beta} (PLC-{beta}) signalling in Drosophila photoreceptors, the phosphatidylinositol transfer protein (PITP) RDGB, is required for lipid transfer at endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (MCS). Depletion of RDGB or its mis-localization away from the ER-PM MCS results in multiple defects in photoreceptor function. Previously, the interaction between the FFAT motif of RDGB and the integral ER protein dVAP-A was shown to be essential for accurate localization to ER-PM MCS. Here, we report that the FFAT/dVAP-A interaction alone is insufficient to localize RDGB accurately; this also requires the function of the C-terminal domains, DDHD and LNS2. Mutations in each of these domains results in mis-localization of RDGB leading to loss of function. While the LNS2 domain is necessary, it is not sufficient for the correct localization of RDGB, which also requires the C-terminal DDHD domain. The function of the DDHD domain is mediated through an intramolecular interaction with the LNS2 domain. Thus, interactions between the additional domains in a multi-domain PITP together lead to accurate localization at the MCS and signalling function.

cell biology↗

bioRxiv2019

The lipid transfer function of RDGB at ER-PM contact sites is regulated by multiple interdomain interactions.

In Drosophila photoreceptors, following Phospholipase C-{beta} activation, the phosphatidylinositol transfer protein (PITP) RDGB, is required to maintain lipid homeostasis at endoplasmic reticulum (ER) plasma membrane (PM) membrane contact sites (MCS). Depletion or mis-localization of RDGB results in multiple defects in photoreceptors. Previously, interaction between the FFAT motif of RDGB with the integral ER protein dVAP-A was shown to be important for its localization at ER-PM MCS. Here, we report that in addition to FFAT motif, a large unstructured region (USR1) of RDGB is required to support the RDGB/dVAP-A interaction. However, interaction with dVAP-A alone is insufficient for accurate localization of RDGB: this also requires association of RDGB with apical PM, through its C-terminal LNS2 domain. Deletion of LNS2 domain results in complete mis-localisation of RDGB and also induces large mis-regulated interdomain movements abrogating RDGB function. Thus, multiple independent interactions between individual domains of RDGB supports its function at ER-PM MCS.

cell biology↗