Biology subjects

Mosalaganti, S.

Publications and source records attributed to Mosalaganti, S..

At least 19 recordsLinked to original sources

bioRxiv2026

The filopodial scaffold polyphosphate dictates cell adhesion-versus-invasion decisions

Inorganic polyphosphate (polyP) is an ancient polymer conserved across all life, serving cell type and location specific functions in every major compartment. Yet its role at the plasma membrane, where it accumulates to peak levels in many primary cells, is largely unknown. Here we identify polyP as a stabilizing component of filopodia, actin based membrane protrusions that govern cell adhesion, contact inhibition, and chemotaxis. Elevating cellular polyP increases filopodial stability and enhances cell adhesion, whereas reducing polyP accelerates filopodial disassembly and promotes cell migration. Mechanistically, we find that polyP acts as a structural filopodial scaffold, recruiting and organizing IRSp53, a membrane curvature inducing protein. We show that metastatic fibroblasts and breast cancer organoids carry markedly reduced and intracellularly reorganized polyP levels relative to their non transformed counterparts. Restoring endogenous polyP via lipid nanoparticle delivery suppresses their invasive phenotypes and reverses prometastatic gene expression signatures, implicating polyP as a primordial tumor suppressor.

cell biology↗

bioRxiv2026

Structural basis of caveolin-driven membrane bending

Caveolins are monotopic membrane proteins essential for caveolae formation and have key roles in signaling and lipid regulation. Caveolins assemble into amphipathic discs with a central {beta}-barrel, an architecture distinct from other membrane-remodeling proteins. These discs embed in the membrane inducing membrane curvature. However, the mechanism of disc-driven bending remains unresolved. Using cryo-electron tomography, structure-guided mutagenesis, and mammalian cell studies, we show that evolutionarily distinct caveolins differ dramatically in their ability to curve membranes despite their conserved architecture. Through computational and theoretical analyses, we demonstrate that patterning of hydrophobic residues along the rim of the disc of human Caveolin-1 induces the deformation of the surrounding leaflet, dictating membrane bending. Finally, we determine a 4.1[A] resolution structure of Caveolin-1 within heterologous caveolae in situ, showing the disc adopts a funnel-like conformation, further shaping membrane architecture. Together, these findings reveal fundamental structural principles that empower caveolins to sculpt and remodel cellular membranes.

cell biology↗

bioRxiv2026

ZCWPW1 organizes telomeric architecture to drive meiotic chromosome movements

Meiotic homolog pairing relies on programmed DNA recombination and large-scale chromosome movements, yet, how these genetic and mechanical events are coordinated remains unclear. ZCWPW1 is a histone reader that recognizes PRDM9-deposited chromatin marks. We identify an unexpected role for ZCWPW1 as a regulator of rapid prophase movements (RPMs). Using super-resolution imaging, we show that ZCWPW1 is strongly enriched at subtelomeric regions of mouse spermatocytes, where it stabilizes TRF1, LINC complex components, dynein, and meiosis-specific cohesin (STAG3). Loss of ZCWPW1 disrupts telomere architecture, weakens telomere-LINC- motor coupling, and abolishes chromosome movement, leading to defective synapsis and pairing, and persistence of DSBs. These defects are more severe than, and mechanistically independent of, those observed in Prdm9-/- spermatocytes. Together, our findings reveal that ZCWPW1 acts independently of PRDM9 as a chromatin-based intranuclear regulator of telomere architecture and telomere-led chromosome movements, thereby linking telomeric chromatin state to nuclear force transmission required for faithful meiotic progression. Significance StatementMeiotic pairing requires recombination and telomere-led chromosome movements, yet no chromatin factor has been shown to regulate both. We identify ZCWPW1 as the first chromatin-based regulator of rapid prophase movements. ZCWPW1 organizes telomeric chromatin and promotes cohesin and motor assembly at telomeres that for force transmission across the nuclear envelope. Loss of ZCWPW1 disrupts the telomere-nuclear envelope mechanical coupling, impairing motion and altering recombination. This function doesnt rely on PRDM9 despite their co-evolution and co-expression, challenging the prevailing view that ZCWPW1 only acts downstream of PRDM9 in DNA repair. Our findings show that chromatin readers can function as structural regulators of genome organization, revealing a conserved mechanism integrating chromosome structure, motion, and repair to ensure proper pairing and fertility.

cell biology↗

bioRxiv2025

A painless nerve growth factor variant uncouples nociceptive and neurotrophic TrkA signaling

Nerve growth factor (NGF) binding to the receptor tyrosine kinase, TrkA, drives neurotrophic signaling essential for neuronal development and survival. This interaction simultaneously drives peripheral pain, making this pathway an attractive but complicated therapeutic target for chronic pain. By integrating single-molecule microscopy, structural and electrophysiology analyses, with a human NGF variant, NGFpainless, which retains neurotrophic effects but abolishes pain, we delineate the molecular mechanisms that bias TrkA signaling towards neurotrophic functions without triggering nociception. We show that, unlike wild-type NGF, NGFpainless fails to sensitize TRPV1 channels to capsaicin, thus disengaging TrkA from the nociceptive pathway. We further show that this selective loss of nociceptive TrkA signaling by NGFpainless results from its reduced ability to activate PLC{gamma}1 and trigger calcium release compared to NGF, while still preserving the ERK and AKT signaling essential for neurotrophic functions. This biased signaling arises from reduced electrostatic complementarity at the TrkA:NGFpainless complex interface, which shortens the lifetime of this functional complex on native membranes. Mutations in TrkA that restore the electrostatic complementarity at the TrkA:NGFpainless interface eliminate biased signaling. This mechanistic understanding of TrkA binding by NGFpainless, and how it differs from NGF, will spur the development of two therapeutic classes of molecules - one that selectively suppresses nociceptive signaling while preserving neurotrophic functions in chronic pain, and another that enhances neurotrophic activity without evoking peripheral pain in neurodegenerative conditions.

neuroscience↗

bioRxiv2025

CTE-Type Tau Filaments in Alzheimer's Disease with Co-morbid LATE-NC

Alzheimers disease (AD), the most common neurodegenerative disease, is defined by {beta}-amyloid plaques and tau neurofibrillary tangles. Tau filaments in AD adopt the "Alzheimers fold", which is distinct from other tauopathies and highly conserved across sporadic and familial AD. However, to date, structural studies have focused on pure AD, despite the high prevalence of comorbid pathologies. In particular, up to half of AD patients harbor limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC). This co-pathology-- consisting of mislocalized and aggregated TDP-43, mainly restricted to the medial temporal lobe--is associated with accelerated clinical decline and more severe tau pathology. Whether there is a structural basis for this clinical synergy remains unknown. Here, using cryo-electron microscopy, we determine the structure of tau filaments from three AD patients with LATE-NC. We show that in addition to the expected Alzheimers fold tau filaments, all cases exhibit a distinct fibril morphology identical to chronic traumatic encephalopathy (CTE) fold tau. Additional sampling revealed CTE neuropathology in one patient, suggestive features without definitive CTE in the second, and no evidence of CTE in the third. These findings raise questions about the relationship between LATE-NC and CTE, the effect of TDP-43 on tau conformation, and the etiology of LATE-NC.

molecular biology↗

bioRxiv2025

Paralogs with distinct phase behaviors broaden the in vivo stress response range of condensates

Paralogs are widespread, but their physiological roles are often masked by redundancy. H-NS, a nucleoid-associated protein in Gram-negative bacteria, typically coexists with paralogs such as StpA, whose functions have remained obscured due to the lack of strong phenotypes. We demonstrate here that the interaction between H-NS and StpA fine-tunes the physico-chemical properties of heterochromatin-forming condensates. H-NS forms dynamic condensates, while StpA assembles into stable insoluble fibrils. Yet together, the two proteins form heterotypic condensates, whose fluidity and stability are tunable by their relative stoichiometry. By increasing the levels of StpA over H-NS, bacteria increase heterochromatin-associated condensate stability, thus preserving gene repression and optimizing bacterial growth under stress conditions. Structural differences at these proteins dimerization sites help explain their distinct phase behaviors. Our findings reveal a novel paradigm in which paralogs that are positioned at the opposite ends of the phase spectrum can fine-tune condensates to promote survival in fluctuating environments.

biochemistry↗

bioRxiv2025

Engineering Spatial Control of Bacterial Organelles

Bacteria were once thought to lack organelles, but it is now clear they confine cellular reactions using an array of membrane- and protein-based compartments. A central question, however, is how bacterial organelles are organized in the cell, and whether their spatial control can be engineered. Here, we show that a two-protein system (McdAB) that positions carboxysomes - CO2-fixing organelles found in autotrophic bacteria - can be repurposed to provide programmable spatial control to diverse organelles in Escherichia coli. McdAB not only restores proper assembly and positioning of heterologously produced carboxysomes in E. coli, but can also be reprogrammed to spatially organize all other known types of bacterial organelles, including encapsulins, biomolecular condensates, and even membrane-bound organelles. Programmable spatial organization of bacterial organelles establishes a new design principle for synthetic biology, where the location of reactions is as tunable as their content. Our work paves the way for more efficient biocatalysis in engineered microbes.

microbiology↗

bioRxiv2025

In situ cryo-ET redefines HPV disassembly and transport paradigms

Human papillomavirus (HPV) causes 4.5% of all human cancers, although a complete cellular basis of infection remains unclear. The mechanisms of virus disassembly and transport are particularly enigmatic. Here, we use in situ cryo-electron tomography (cryo-ET) to provide direct visualization of high-risk HPV16 during its infection cycle. We demonstrate that, when HPV reaches the lysosome, it remains intact and infection competent, contrary to the prevailing view of viral inactivation within lysosomes. We challenge the current model of HPV trafficking, which predicts progressive disassembly of the L1 capsid during retrograde transport, by showing that HPV remains intact when it reaches the Golgi en route to the nucleus for infection. Finally, we provide snapshots of the HPV containing transport vesicles budding from the Golgi lumen in a COPI coat-dependent fashion and show that HPV is connected to the overlying membrane by proteinaceous structural bridges, possibly formed by the L2 capsid, to guide virus trafficking. Our results redefine the dominant models of HPV entry, revealing the virus capsids resilience to disassembly during retrograde trafficking, a coat-dependent budding mechanism for transport, and demonstrate that the lysosome serves as a reservoir for infectious HPV.

cell biology↗

bioRxiv2025

Insights into the structure and modulation of human TWIK-2

The Tandem of pore domain in a Weak Inward Rectifying K+ channel 2 (TWIK-2; KCNK6) is a member of the Two-Pore Domain K+ (K2P) channel family, which is associated with pulmonary hypertension, lung injury, and inflammation. The structure and regulatory mechanisms of TWIK-2 remain largely unknown. Here, we present the cryo-electron microscopy (cryo-EM) structure of human TWIK-2 at [~]3.7 [A] and highlight its conserved and unique features. Using automated whole-cell patch clamp recordings, we demonstrate that gating in TWIK-2 is voltage-dependent and insensitive to changes in the extracellular pH. We identify key residues that influence TWIK-2 activity by employing structure and sequence-guided site-directed mutagenesis and provide insights into the possible mechanism of TWIK-2 regulation. Additionally, we demonstrate the application of high-throughput automated whole-cell patch clamp platforms to screen small molecule modulators of TWIK-2. Our work serves as a foundation for designing high-throughput small molecule screening campaigns to identify specific high-affinity TWIK-2 modulators, including promising new anti-inflammatory therapeutics.

biophysics↗

bioRxiv2024

Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and alter their functional properties

The human genome is replete with sequences derived from foreign elements including endogenous retrovirus-like proteins of unknown function. Here we show that UBQLN2, a ubiquitin-proteasome shuttle factor implicated in neurodegenerative diseases, is regulated by the linked actions of two retrovirus-like proteins, RTL8 and PEG10. RTL8 confers on UBQLN2 the ability to complex with and regulate PEG10. PEG10, a core component of stress granules, drives the recruitment of UBQLN2 to stress granules under various stress conditions, but can only do so when RTL8 is present. Changes in PEG10 levels further remodel the kinetics of stress granule disassembly and overall composition by incorporating select extracellular vesicle proteins. Within stress granules, PEG10 forms virus-like particles, underscoring the structural heterogeneity of this class of biomolecular condensates. Together, these results reveal an unexpected link between pathways of cellular proteostasis and endogenous retrovirus-like proteins.

neuroscience↗

bioRxiv2024

Molecular Visualization of Neuronal TDP43 Pathology In Situ

Nuclear exclusion and cytoplasmic accumulation of the RNA-binding protein TDP43 are characteristic of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Despite this, the origin and ultrastructure of cytosolic TDP43 deposits remain unknown. Accumulating evidence suggests that abnormal RNA homeostasis can drive pathological TDP43 mislocalization, thereby enhancing RNA misprocessing due to the loss of nuclear TDP43, and engendering a cycle that ultimately leads to cell death. Here, we demonstrate that the addition of small monovalent oligonucleotides successfully recapitulates pathological TDP43 mislocalization and aggregation, aberrant splicing, and degeneration in iPSC-derived neurons (iNeurons). By employing a tailored multimodal in situ cryo-correlative light and electron microscopy pipeline, we examine the localization and aggregation of TDP43 in near-native conditions. We discover that mislocalized TDP43 accumulates and forms ordered fibrils within autophagosomes and lysosomes in iNeurons, as well as in ALS/FTLD patient tissue. We provide the first high-resolution snapshots of TDP43 aggregates in situ, delivering an unprecedented view of the earliest pathogenic events underlying ALS, FTLD, and related TDP43 proteinopathies.

neuroscience↗

bioRxiv2024

Neutrophils secrete exosome-associated DNA to resolve sterile acute inflammation

Acute inflammation, characterized by a rapid influx of neutrophils, is a protective response that can lead to chronic inflammatory diseases when left unresolved. Secretion of LTB4-containing exosomes is required for effective neutrophil infiltration during inflammation. In this study, we show that neutrophils release nuclear DNA in a non-lytic, rapid, and repetitive manner, via a mechanism distinct from suicidal NET release and cell death. The packaging of nuclear DNA occurs in the lumen of nuclear envelope (NE)-derived multivesicular bodies (MVBs) that harbor the LTB4 synthesizing machinery and is mediated by the lamin B receptor (LBR) and chromatin decondensation. Disruption of secreted exosome-associated DNA (SEAD) in a model of sterile inflammation in mouse skin amplifies and prolongs the presence of neutrophils, impeding the onset of resolution. Together, these findings advance our understanding of neutrophil functions during inflammation and the physiological significance of NETs, with implications for novel treatments for inflammatory disorders.

immunology↗

bioRxiv2024

Rapid structural analysis of bacterial ribosomes in situ

Rapid structural analysis of purified proteins and their complexes has become increasingly common thanks to key methodological advances in cryo-electron microscopy (cryo-EM) and associated data processing software packages. In contrast, analogous structural analysis in cells via cryo-electron tomography (cryo-ET) remains challenging due to critical technical bottlenecks, including low-throughput sample preparation and imaging, and laborious data processing methods. Here, we describe the development of a rapid in situ cryo-ET sample preparation and data analysis workflow that results in the routine determination of sub-nm resolution ribosomal structures. We apply this workflow to E. coli, producing a 5.8 [A] structure of the 70S ribosome from cells in less than 10 days, and we expect this workflow will be widely applicable to related bacterial samples.

cell biology↗

bioRxiv2024

BRD4-mediated epigenetic regulation of endoplasmic reticulum-mitochondria contact sites is governed by the mitochondrial complex III

Inter-organellar communication is critical for cellular metabolic homeostasis. One of the most abundant inter-organellar interactions are those at the endoplasmic reticulum and mitochondria contact sites (ERMCS). However, a detailed understanding of the mechanisms governing ERMCS regulation and their roles in cellular metabolism are limited by a lack of tools that permit temporal induction and reversal. Through unbiased screening approaches, we identified fedratinib, an FDA-approved drug, that dramatically increases ERMCS abundance by inhibiting the epigenetic modifier BRD4. Fedratinib rapidly and reversibly modulates mitochondrial and ER morphology and alters metabolic homeostasis. Moreover, ERMCS modulation depends on mitochondria electron transport chain complex III function. Comparison of fedratinib activity to other reported inducers of ERMCS revealed common mechanisms of induction and function, providing clarity and union to a growing body of experimental observations. In total, our results uncovered a novel epigenetic signaling pathway and an endogenous metabolic regulator that connects ERMCS and cellular metabolism.

cell biology↗

bioRxiv2023

Structure of the human systemic RNAi defective transmembrane protein 1 (hSIDT1) reveals the conformational flexibility of its lipid binding domain

In C. elegans, inter-cellular transport of the small non-coding RNA causing systemic RNA interference (RNAi) is mediated by the transmembrane protein SID1, encoded by the sid1 gene in the systemic RNA interference-defective (sid) loci. SID1 shares structural and sequence similarity with cholesterol uptake protein 1 (CHUP1) and is classified as a member of the cholesterol uptake family (ChUP). Although systemic RNAi is not an evolutionarily conserved process, the sid gene products are found across the animal kingdom, suggesting the existence of other novel gene regulatory mechanisms mediated by small non-coding RNAs. Human homologs of sid gene products - hSIDT1 and hSIDT2 - mediate contact-dependent lipophilic small non-coding dsRNA transport. Here, we report the structure of recombinant human SIDT1. We find that the extra- cytosolic domain (ECD) of hSIDT1 adopts a double jelly roll fold, and the transmembrane domain (TMD) exists as two modules - a flexible lipid binding domain (LBD) and a rigid TMD core. Our structural analyses provide insights into the inherent conformational dynamics within the lipid binding domain in cholesterol uptake (ChUP) family members.

biophysics↗

bioRxiv2023

Structure of the human heparan-α-glucosaminide N-acetyltransferase (HGSNAT)

Degradation of heparan sulfate (HS), a glycosaminoglycan (GAG) comprised of repeating units of N-acetylglucosamine and glucuronic acid, begins in the cytosol and is completed in the lysosomes. Acetylation of the terminal non-reducing amino group of -D-glucosamine of HS is essential for its complete breakdown into monosaccharides and free sulfate. Heparan--glucosaminide N-acetyltransferase (HGSNAT), a resident of the lysosomal membrane, catalyzes this essential acetylation reaction by accepting and transferring the acetyl group from cytosolic acetyl-CoA to terminal -D-glucosamine of HS in the lysosomal lumen. Mutation-induced dysfunction in HGSNAT causes abnormal accumulation of HS within the lysosomes and leads to an autosomal recessive neurodegenerative lysosomal storage disorder called mucopolysaccharidosis IIIC (MPS IIIC). There are no approved drugs or treatment strategies to cure or manage the symptoms of, MPS IIIC. Here, we use cryo-electron microscopy (cryo-EM) to determine a high-resolution structure of the HGSNAT-acetyl-CoA complex, the first step in HGSNAT catalyzed acetyltransferase reaction. In addition, we map the known MPS IIIC mutations onto the structure and elucidate the molecular basis for mutation-induced HGSNAT dysfunction.

biophysics↗

bioRxiv2021

Artificial intelligence reveals nuclear pore complexity

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport. Their intricate 120 MDa architecture remains incompletely understood. Here, we report a near-complete structural model of the human NPC scaffold with explicit membrane and in multiple conformational states. We combined AI-based structure prediction with in situ and in cellulo cryo-electron tomography and integrative modeling. We show that linker Nups spatially organize the scaffold within and across subcomplexes to establish the higher-order structure. Microsecond-long molecular dynamics simulations suggest that the scaffold is not required to stabilize the inner and outer nuclear membrane fusion, but rather widens the central pore. Our work exemplifies how AI-based modeling can be integrated with in situ structural biology to understand subcellular architecture across spatial organization levels. One sentence summaryAn AI-based, dynamic model of the human nuclear pore complex reveals how the protein scaffold and the nuclear envelope are coupled inside cells.

molecular biology↗

bioRxiv2020

3D super-resolution fluorescence microscopy maps the variable molecular architecture of the Nuclear Pore Complex

Nuclear pore complexes (NPCs) are large macromolecular machines that mediate the traffic between the nucleus and the cytoplasm. In vertebrates, each NPC consists of ~1000 proteins, termed nucleoporins, and has a mass of over 100 MDa. While a pseudo-atomic static model of the central scaffold of the NPC has recently been assembled by integrating data from isolated proteins and complexes, many structural components still remain elusive due to the enormous size and flexibility of the NPC. Here, we explored the power of 3D super-resolution microscopy combined with computational classification and averaging to explore the 3D structure of the NPC in single human cells. We show that this approach can build the first integrated 3D structural map containing both central as well as peripheral NPC subunits with molecular specificity and nanoscale resolution. Our unbiased classification of over ten thousand individual NPCs indicates that the nuclear ring and the nuclear basket can adopt different conformations. Our approach opens up the exciting possibility to relate different structural states of the NPC to function in situ.

cell biology↗