Biology subjects

Balachandra, S.

Publications and source records attributed to Balachandra, S..

5 recordsLinked to original sources

bioRxiv2024

Visualizing developmental dynamics of nuclear morphology and transport machinery in Drosophila

Communication between the cytoplasm and the nucleus requires a continuous exchange of molecules across the nuclear envelope (NE). The nuclear pore complex (NPC) is the gateway embedded in the NE through which cargo moves, while transport receptors mediate the passage of macromolecules through the NPC. Although their essential role as the components of the nuclear transport machinery has been extensively studied, how these factors respond to developmental and environmental cues has been underexplored. Here we tag the nucleoporin Nup96 and the transport receptor Imp{beta} with mEGFP and mScarlet-I at their endogenous loci in Drosophila. We demonstrate the functionality of these markers in multiple tissues and offer new options for better visualization of nuclear morphology in densely packed, complex tissues. Then, we characterize the spatiotemporal dynamics of these markers in multiple developmental contexts. We find that Nup96 and Imp{beta} form cytoplasmic puncta, whose size, numbers, and co-localization patterns change dynamically during oogenesis and early embryogenesis. Moreover, we find that the abundance of NPCs per nucleus decreases during early embryogenesis, complementing the emerging model in which NPCs play a regulatory role in development. The tools and observations described here will be useful in understanding the dynamic regulation of nuclear morphology and transport machinery in development.

cell biology↗

bioRxiv2024

RIPK1 is essential for Herpes Simplex Virus-triggered ZBP1-dependent necroptosis in human cells

Necroptosis initiated by the host sensor Z-NA Binding Protein-1 (ZBP1) is essential for host defense against a growing number of viruses, including Herpes Simplex Virus-1 (HSV-1). Studies with HSV-1 and other necroptogenic stimuli in murine settings have suggested that ZBP1 triggers necroptosis by directly complexing with the kinase RIPK3. Whether this is also the case in human cells, or whether additional co-factors are needed for ZBP1-mediated necroptosis, is unclear. Here, we show that ZBP1-induced necroptosis in human cells requires RIPK1. We have found that RIPK1 is essential for forming a stable and functional ZBP1-RIPK3 complex in human cells, but is dispensable for the formation of the equivalent murine complex. The RIP Homology Interaction Motif (RHIM) in RIPK3 is responsible for this difference between the two species, because replacing the RHIM in human RIPK3 with the RHIM from murine RIPK3 is sufficient to overcome the requirement for RIPK1 in human cells. These observations describe a critical mechanistic difference between mice and humans in how ZBP1 engages in necroptosis, with important implications for treating human diseases.

cell biology↗

bioRxiv2019

Bellymount enables longitudinal, intravital imaging of abdominal organs and the gut microbiota in adult Drosophila

Cell- and tissue-level processes often occur across days or weeks, but few imaging methods can capture such long timescales. Here we describe Bellymount, a simple, non-invasive method for longitudinal imaging of the Drosophila abdomen at sub-cellular resolution. Bellymounted flies remain live and intact, so the same individual can be imaged serially to yield vivid time series of multi-day processes. This feature opens the door to longitudinal studies of Drosophila internal organs in their native context. Exploiting Bellymounts capabilities, we track intestinal stem cell lineages and gut microbial colonization in single flies, revealing spatiotemporal dynamics undetectable by previously available methods.

developmental biology↗

bioRxiv2018

Long-term live imaging of the Drosophila adult midgut reveals real-time dynamics of cell division, differentiation, and loss

Organ renewal is governed by the dynamics of cell division, differentiation, and loss. To study these dynamics in real time, here we present a platform for extended live imaging of the adult Drosophila midgut, a premier genetic model for stem cell-based organs. A window cut into a living animal allows the midgut to be imaged while intact and physiologically functioning. This approach prolongs imaging sessions to 12-16 hours and yields movies that document cell and tissue dynamics at vivid spatiotemporal resolution. Applying a pipeline for movie processing and analysis, we uncover new, intriguing cell behaviors: that mitotic stem cells dynamically re-orient, that daughter cells delay fate-determining Notch activation for many hours after birth, and that enterocytes extrude via constriction of a pulsatile cadherin ring. By enabling real-time study of cellular phenomena that were previously inaccessible, our platform opens a new realm for dynamic understanding of midgut organ renewal.

cell biology↗

bioRxiv2017

Feedback regulation of steady-state epithelial turnover and organ size

Epithelial organs undergo steady-state turnover throughout adult life, with old cells being continually replaced by the progeny of stem cell divisions1. To avoid hyperplasia or atrophy, organ turnover demands strict equilibration of cell production and loss2-4. However, the mechanistic basis of this equilibrium is unknown. Using the adult Drosophila intestine5, we find that robustly precise turnover arises through a coupling mechanism in which enterocyte apoptosis breaks feedback inhibition of stem cell divisions. Healthy enterocytes inhibit stem cell division through E-cadherin, which prevents secretion of mitogenic EGFs by repressing transcription of the EGF maturation factor rhomboid. Individual apoptotic enterocytes promote divisions by loss of E-cadherin, which releases cadherin-associated {beta}-catenin/Armadillo and p120-catenin to induce rhomboid. Induction of rhomboid in the dying enterocyte triggers EGFR activation in stem cells within a discrete radius. When we block apoptosis, E-cadherin-controlled feedback suppresses divisions, and the organ retains the same number of cells. When we disrupt feedback, apoptosis and divisions are uncoupled, and the organ develops either hyperplasia or atrophy. Altogether, our work demonstrates that robust cellular balance hinges on the obligate coupling of divisions to apoptosis, which limits the proliferative potential of a stem cell to the precise time and place that a replacement cell is needed. In this manner, localized cell-cell communication gives rise to tissue-level homeostatic equilibrium and constant organ size.

developmental biology↗