Biology subjects

Saminathan, A.

Publications and source records attributed to Saminathan, A..

9 recordsLinked to original sources

bioRxiv2026

Naked antisense oligonucleotides remain endolysosomally sequestered despite induced membrane damage

Antisense oligonucleotides (ASOs) enter cells efficiently, but the compartment from which productive escape occurs remains uncertain. We used live-cell microscopy, ratiometric pH measurements and 3D focused ion beam scanning electron microscopy (FIB-SEM) in U2OS cells to track a Malat1-targeting ASO from uptake to delivery. The ASO entered by endocytosis and accumulated in late endosomes, endolysosomes and lysosomes, where it induced luminal neutralization without galectin-3 recruitment or limiting-membrane rupture. Under conditions that reduced Malat1-RNA by >90%, quantitative imaging showed that less than 4% of internalized ASOs reached the nucleus. L-leucyl-L-leucine methyl ester (LLOMe)-induced membrane damage released co-internalized dextran but not ASOs, showing that ASOs remain sequestered even in damaged late endocytic compartments. In apilimod-expanded organelles, ASOs concentrated at limiting membranes and intraluminal foci with constrained motion, consistent with association with membrane and luminal structures. Although G3BP1/2 has been proposed to plug damaged endocytic membranes, we detected no recruitment of G3BP1 to endosomes or lysosomes; loss of G3BP1 and G3BP2 increased functional delivery modestly. We therefore propose that productive escape occurs earlier in endocytosis, most likely in early or recycling endosomes, where ASOs would still be unbound within the lumen and where membrane fusion and fission could generate perforations permitting release.

cell biology↗

bioRxiv2025

How endosomal PIKfyve inhibition prevents viral membrane fusion and entry

Enveloped viruses enter cells by membrane fusion. The viral membrane fuses with a host membrane, either at the cell surface or within endocytic compartments. For endocytic entry, fusion is typically triggered by low pH and often requires proteolytic priming by compartment-specific host proteases, which together define the site and mechanism of fusion and shape viral tropism. Inhibition of the lipid kinase PIKfyve, which generates PI(5)P and PI(3,5)P2 in late endosomes and lysosomes, swells those compartments and blocks infection by a subset of enveloped viruses, including Ebola virus, Marburg virus, coronaviruses (SARS-CoV-2), and VSV chimeras bearing Ebola, SARS-CoV-2, or Lassa glycoproteins, while showing minor effects on H1N1 influenza and no effect on VSV or VSV-rabies chimeras. In the work reported here, we have determined the basis for selectivity. We show that swelling of late endosomes/lysosomes, independent of changes in lipid composition or altered virion trafficking, is sufficient to block virus-endosome fusion and genome release, even when endosomal acidity is preserved. Acute PIKfyve inhibition with apilimod or brief hypotonic treatment produced endosomal swelling and impaired infection by interrupting a late endosomal entry step. Live-cell 3D lattice light-sheet fluorescence microscopy imaging tracked fluorescent virions accumulating and arresting in late endosomes prior to fusion, and single-cell, single-round assays confirmed loss of infectivity. These data support a simple biophysical mechanism: endo-lysosomal swelling, likely increasing endosomal membrane tension, creates an energy barrier to fusion and genome release. Inducing such swelling may offer a general strategy to inhibit viruses that depend on late endosomal entry. SIGNIFICANCE STATEMENTWhy does inhibiting the endosomal lipid kinase PIKfyve, which generates PI(5)P and PI(3,5)P2, block entry of some enveloped viruses but spare VSV-G? Using single-round infectivity and live-cell 3D imaging, we show that acute PIKfyve inhibition traps incoming VSV chimeras bearing SARS-CoV-2 spike or Ebola GP in swollen late endosomes/lysosomes before fusion and genome release. Hypotonic swelling phenocopies the block, and removing glutamine prevents apilimod-induced swelling and largely restores infection, arguing that swelling--not altered phosphoinositides--restricts entry. We propose a physical mechanism: increased endosomal membrane tension raises the energetic barrier for fusion-pore formation and genome release. Modulating endolysosomal volume may therefore inhibit viruses that rely on late endosomal entry.

cell biology↗

bioRxiv2024

Constitutive Endolysosomal Perforation in Neurons allows Induction of alpha-Synuclein Aggregation by Internalized Pre-Formed Fibrils

The endocytic pathway is both an essential route of molecular uptake in cells and a potential entry point for pathology-inducing cargo. The cell-to-cell spread of cytotoxic aggregates, such as those of -synuclein (-syn) in Parkinsons Disease (PD), exemplifies this duality. Here we used a human iPSC-derived induced neuronal model (iNs) prone to death mediated by aggregation in late endosomes and lysosomes of endogenous -syn, seeded by internalized pre-formed fibrils of -syn (PFFs). This PFF-mediated death was not observed with parental iPSCs or other non-neuronal cells. Using live-cell optical microscopy to visualize the read out of biosensors reporting endo-lysosome wounding, we discovered that up to about 10% of late endosomes and lysosomes in iNs exhibited spontaneous constitutive perforations, regardless of the presence of internalized PFFs. This wounding, absent in parental iPSCs and non-neuronal cells, corresponded to partial damage by nanopores in the limiting membranes of a subset of endolysosomes directly observed by volumetric focused ion beam scanning electron microscopy (FIB-SEM) in iNs and in CA1 pyramidal neurons from mouse brain, and not found in iPSCs or in other non-neuronal cells in culture or in mouse liver and skin. We suggest that the compromised limiting membranes in iNs and neurons in general are the primary conduit for cytosolic -syn to access PFFs entrapped within endo-lysosomal lumens, initiating PFF-mediated -syn aggregation. Significantly, eradicating the intrinsic endolysosomal perforations in iNs by inhibiting the endosomal Phosphatidylinositol-3-Phosphate/Phosphatidylinositol 5-Kinase (PIKfyve kinase) using Apilimod or Vacuolin-1 markedly reduced PFF-induced -syn aggregation, despite PFFs continuing to enter the endolysosomal compartment. Crucially, this intervention also diminished iN death associated with PFF incubation. Our results reveal the surprising presence of intrinsically perforated endo-lysosomes in neurons, underscoring their crucial early involvement in the genesis of toxic -syn aggregates induced by internalized PFFs. This discovery offers a basis for employing PIKfyve kinase inhibition as a potential therapeutic strategy to counteract synucleinopathies.

cell biology↗

bioRxiv2023

Deep learning assisted single particle tracking for automated correlation between diffusion and function

Sub-cellular diffusion in living systems reflects cellular processes and interactions. Recent advances in optical microscopy allow the tracking of this nanoscale diffusion of individual objects with an unprecedented level of precision. However, the agnostic and automated extraction of functional information from the diffusion of molecules and organelles within the sub-cellular environment, is labor-intensive and poses a significant challenge. Here we introduce DeepSPT, a deep learning framework to interpret the diffusional 2D or 3D temporal behavior of objects in a rapid and efficient manner, agnostically. Demonstrating its versatility, we have applied DeepSPT to automated mapping of the early events of viral infections, identifying distinct types of endosomal organelles, and clathrin-coated pits and vesicles with up to 95% accuracy and within seconds instead of weeks. The fact that DeepSPT effectively extracts biological information from diffusion alone indicates that besides structure, motion encodes function at the molecular and subcellular level.

biophysics↗

bioRxiv2023

TMEM165 acts as a proton-activated Ca2+ importer in lysosomes

Lysosomal calcium (Ca2+) release is critical to cell signaling and is mediated by well-known lysosomal Ca2+ channels. Yet, how lysosomes refill their Ca2+ remains hitherto undescribed. Here, from an RNAi screen, we identify an evolutionarily conserved gene, lci-1, that facilitates lysosomal Ca2+ entry in C. elegans and in mammalian cells. Its human homolog TMEM165, previously designated as a Golgi-resident Ca2+/H+ exchanger (CAX), has a minor lysosomal population of unknown function. Using genetics, lysosomal Ca2+ imaging and electrophysiology, we show that TMEM165 acts as a proton-activated, lysosomal Ca2+ importer in lysosomes. Defects in lysosomal Ca2+ channels cause several neurodegenerative diseases, and knowledge of lysosomal Ca2+ importers may provide new avenues to explore the physiology of Ca2+ channels.

cell biology↗

bioRxiv2022

Humanized antibody potently neutralizes all SARS-CoV-2 variants by a novel mechanism

SARS-CoV-2 Omicron variants have generated a world-wide health crisis due to resistance to most approved SARS-CoV-2 neutralizing antibodies and evasion of antibodies induced by vaccination. Here, we describe the SARS-CoV-2 neutralizing SP1-77 antibody that was generated from a humanized mouse model with a single human VH1-2 and V{kappa}1-33-associated with immensely diverse complementarity-determining-region-3 (CDR3) sequences. SP1-77 potently and broadly neutralizes SARS-CoV-2 variants of concern and binds the SARS-CoV-2 spike protein receptor-binding-domain (RBD) via a novel-CDR3-based mode. SP1-77 does not block RBD-binding to the ACE2-receptor or endocytosis step of viral entry, but rather blocks membrane fusion. Our findings provide the first mechanistic insight into how a non-ACE2 blocking antibody potently neutralizes SARS-CoV-2, which may inform strategies for designing vaccines that robustly neutralize current and future SARS-CoV-2 variants.

immunology↗

bioRxiv2022

Tracking infectious entry routes of SARS-CoV-2

SARS-CoV-2 cell entry starts with membrane attachment and ends with spike-protein (S) catalyzed membrane fusion depending on two cleavage steps, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time 3D single virion tracking, we show fusion and genome penetration requires virion exposure to an acidic milieu of pH 6.2-6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2 overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2 expressing cells in the acidic milieu of the nasal cavity. Significance StatementInfection by SARS-CoV-2 depends upon the S large spike protein decorating the virions and is responsible for receptor engagement and subsequent fusion of viral and cellular membranes allowing release of virion contents into the cell. Using new single particle imaging tools, to visualize and track the successive steps from virion attachment to fusion, combined with chemical and genetic perturbations of the cells, we provide the first direct evidence for the cellular uptake routes of productive infection in multiple cell types and their dependence on proteolysis of S by cell surface or endosomal proteases. We show that fusion and content release always require the acidic environment from endosomes, preceded by liberation of the S1 fragment which depends on ACE2 receptor engagement. One sentence summaryDetailed molecular snapshots of the productive infectious entry pathway of SARS-CoV-2 into cells

microbiology↗

bioRxiv2020

Tubular lysosomes harbor active ion gradients and poise macrophages for phagocytosis

Lysosomes adopt dynamic, tubular states that regulate antigen presentation, phagosome resolution and autophagy. To date, tubular lysosomes have been studied either by inducing autophagy or by activating immune cells, both of which lead to cell states where lysosomal gene expression differs from the resting state. Therefore, it has been challenging to pinpoint the specific biochemical properties lysosomes acquire upon tubulation that could drive their functionality. We describe a DNA-based assembly that tubulates lysosomes in macrophages without activating them. Lumenal proteolytic activity maps at single lysosome resolution revealed that tubular lysosomes were less degradative. Further, they showed striking proximal to distal lumenal pH and Ca2+ gradients. Such gradients had been predicted, but never previously observed. We now identify a role for tubular lysosomes whereby they poise resting macrophages for phagocytosis. The ability to tubulate lysosomes without having to starve or activate immune cells may help reveal new roles for tubular lysosomes.

cell biology↗

bioRxiv2019

A DNA-based voltmeter for organelles

The role of membrane potential in most intracellular organelles remains unexplored because of the lack of suitable probes. We describe a DNA-based fluorescent reporter that quantitates membrane potential and can be targeted to specific organelles in live cells. It is equipped with a voltage sensitive fluorophore, a reference fluorophore for ratiometric quantification, and acts as an endocytic tracer. We could thereby measure the membrane potential of different intracellular organelles in living cells, which has not been possible previously. Our understanding of how membrane potential regulates organelle biology is poised to expand through the use of these new sensors.\n\nOne Sentence SummaryUsing a DNA-based voltmeter we can non-invasively measure the membrane potential of specific organelles in live cells.

biophysics↗