Biology subjects

Pucadyil, T.

Publications and source records attributed to Pucadyil, T..

11 recordsLinked to original sources

bioRxiv2026

Dynamin-2 is a fission factor for SNX1-coated endosomal tubules

Sorting nexins of the SNX-BAR family shape endosomal membranes into tubular carriers that route cargo away from the degradative pathway, but we poorly understand the machinery that cuts these tubules to release transport carriers. Here we show that Dynamin-2 (Dyn2) is a fission factor for SNX1-coated tubules. In HeLa cells, Dyn2 localises to SNX1 tubules, and its knockout lengthens them and produces interconnected networks. Dyn2 binds SNX1 directly, and this interaction requires both the proline-rich domain of Dyn2 and the N-terminus of SNX1. On supported lipid bilayers, SNX1 alone tubulates membranes, and adding Dyn2 and GTP converts the emergent tubules into vesicles, indicating that no additional factors are needed for this activity. In cells, loss of Dyn2 redirects the retromer cargo GLUT1 to a LAMP1-positive compartment and reduces glucose uptake. These findings identify Dyn2 as the enzyme that releases SNX1 carriers and link this scission step to cargo recycling.

cell biology↗

bioRxiv2026

Phosphoinositides protonation states dictate AKT1 binding with membrane

Phosphoinositides are key regulators of membrane-associated signaling, yet their electrostatic properties are commonly represented by a single nominal charge in molecular simulations and structural models. Using the essential protein kinase AKT1 as a model system, we show that phosphoinositide protonation microstates, both the degree and positional placement of protons, govern peripheral membrane-protein recognition. By integrating quantum-mechanics-derived headgroup parameters, microsecond-scale all-atom molecular dynamics, and umbrella-sampling free-energy calculations with the available solid-state NMR-derived lipid populations via Bayesian/Maximum Entropy reweighting, we account explicitly for the thermodynamic ensemble of phosphoinositide proto-nation states. This population-weighted free-energy framework provides a quantitative mechanistic rationale for the strict specificity of wild-type AKT1 toward PI(3, 4, 5)P3 and elucidates how the oncogenic sentry mutation (E17K) alters this specificity to enable high-affinity PIP2 binding. Furthermore, Proximity-based Labeling of Membrane Associated Proteins (PLiMAP) assays experimentally confirm that AKT1 pleckstrin homology domain binding to distinct phosphoinositide species is differentially sensitive to pH. Together, our findings establish a direct connection between phosphoinositide protonation equilibria, binding energetics, and AKT1 lipid specificity. Beyond AKT1, these results demonstrate that the dynamic protonation microstates of anionic phospholipids act as a functional regulatory layer governing peripheral membrane-protein recruitment across diverse cellular microenvironments.

biophysics↗

bioRxiv2026

Division-arrest induced filamentation protects uropathogenic Escherichia coli from killing by the cathelicidin antimicrobial peptide LL-37

Antimicrobial peptides are a key component of the innate immune system, serving both as signaling and immunomodulatory molecules and also as direct antimicrobials toward numerous bacterial species. One of the key antimicrobial peptides in humans is LL-37 but the mechanism of bacterial killing remains poorly characterized. Using live-cell imaging, we observe that LL-37 preferentially targets actively dividing E. coli cells. Permeabilization occurs during late-stage division at the bacterial septum with both of the newly dividing cells being killed at the same time. Death occurs downstream of the recruitment and dissembly of FtsZ and FtsN and concomitantly with AmiB association at the divisome, consistent with killing occurring during late division. Model membrane tubes that topologically mimic the late-stage cytoplasmic membrane intermediates during division also undergo rapid size- and lipid composition-dependent fission, which likely reflect the ability of LL-37 to disrupt membranes. Intriguingly, we show that inducing bacterial filamentation results in resistance to LL-37 mediated killing. Resistance to LL-37 appears to be a general feature of bacterial filamentation as overexpression of queE, sulA or damX all resulted in reduced killing by LL-37. As filamentation is often observed during UPEC infection, we hypothesize that filamentation associated resistance to host innate immunity may be a key feature of UPEC pathogenesis that protects bacteria during extrusion from infection bladder epithelial cells, when exposure to soluble and cellular host defences is likely highest. This observation may provide an important avenue for therapeutic intervention. SignificanceAntimicrobial peptides are a key component of innate immunity. The human cathelicidin peptide, LL-37, is membrane active but the mechanism of sensitization in living systems is poorly understood. We show that dividing E. coli cells are highly susceptible to LL-37 mediated disruption. Killing occurs downstream of the assembly and activation of functional divisomes. In vitro membrane nanotubes also demonstrate sensitivity to LL-37 in a lipid composition and diameter-dependent manner. Cells with stalled division are protected from LL-37. Urinary tract infections caused by uropathogenic E. coli (UPEC) are a major cause of morbidity globally. UPEC form filamentous bacteria late in the human infection cycle. UPEC filaments are protected from LL-37 killing and suggest that interfering with bacterial filamentation might potentiate innate immune control of UTIs.

microbiology↗

bioRxiv2025

Membrane remodeling by the Bacterial Dynamin-like Protein (BDLP) from Nostoc punctiforme

Membranes are fundamental to biological systems as they serve as barriers to compartmentalize the cell and its cytoplasm. They are constantly remodeled in shape and composition during the formation and maintenance of organelles. The large GTPase dynamins are well characterized for membrane shape remodeling in eukaryotes but their functions in prokaryotes are less characterized. Here, we determine the lipid-binding, enzymatic and membrane remodeling activities of the Bacterial Dynamin-Like Protein (BDLP) from the cyanobacterium Nostoc punctiforme. We find that BDLP binds phosphatidyl glycerol (PG)-containing membranes, but unlike other soluble dynamins, shows little stimulation in GTPase activity. This enzymatic feature is like that seen among the fusion dynamins to which BDLP shows structural similarities. FRET-based bulk vesicle fusion assays however reveal that BDLP is incapable of membrane fusion. To further understand BDLP functions, we turned to microscopic analysis of BDLP on membranes templates. We find that the GTP-bound BDLP forms a rigid scaffold that is capable of bending and imposing a defined curvature. In contrast, the GDP-bound state is loosely organized and unable to remodel membrane shape. Our results indicate that BDLP intrinsically functions as a protein scaffold whose organization is regulated by nucleotide binding.

biochemistry↗

bioRxiv2025

Vacuolar protein sorting-associated protein 1 (Vps1) has membrane constricting and severing abilities required for endosomal protein trafficking

Endosomal protein trafficking pathways are fundamental to maintaining organelle identity and function and are conserved from yeast to humans. The retromer pathway traffics cargo from endosomes to the trans Golgi network and defects in this pathway are linked to a variety of metabolic and neurological disorders. The yeast dynamin Vacuolar protein sorting-associated protein 1 (Vps1) functions in the retromer pathway. Vps1 has been found to associate with tubular endosomes undergoing severing, implying a role in membrane fission. Current biochemical and structural analyses of Vps1 however seem to indicate that it is not sufficient for fission. Here, we analyze Vps1 functions using cell-free reconstitution, live cell imaging and proteomics of isolated yeast vacuoles. We find that the endosomal lipid phosphatidylinositol-3-phosphate (PI(3)P) stabilizes Vps1 to form protein scaffolds around tubular membrane substrates. GTP hydrolysis forces constriction of tubules to a critical [~]7 nm radius, ultimately causing their fission. Furthermore, we discover that the Insert B (InsB) in Vps1 harbor conserved lysine- rich (3K) lipid-binding and phenylalanine-rich (4F) self-assembly motifs. Mutating these motifs renders Vps1 incapable of membrane fission and abrogates its functions in endosomal protein sorting. Quantitative vacuolar proteomic analysis reveals that loss of Vps1 results in the enrichment of a small set of proteins, most of which represent retromer cargo while causing depletion of the bulk of proteins in the vacuole. Vps1 functions in endosomal protein sorting are therefore critical for the global regulation of the vacuole proteome.

biochemistry↗

bioRxiv2024

Wag31, a membrane tether, is crucial for lipid homeostasis in mycobacteria

The mycobacterial cytoskeletal protein Wag31 is necessary for maintaining cell shape and directing cellular growth and elongation. Wag31 has a characteristic N-terminal DivIVA-domain and a C-terminal coiled-coil domain. While the role of Wag31 in polar elongation is known, there is limited mechanistic insight on how it orchestrates growth and elongation. In this report, we delineate roles of the N- and C-terminal domains of Wag31 using genetics, state-of-the-art multi-omics, biochemical, and imaging approaches. We show that Wag31 predominantly interacts with several membrane-associated proteins involved in lipid metabolism, cell wall synthesis and division. Native levels of Wag31 are critical for the maintenance and distribution of membrane lipids. Both depletion and overexpression of Wag31 perturbs lipid homeostasis, leading to the formation of intracellular lipid inclusions (ILIs). Protein-lipid crosslinking and imaging studies reveal that purified Wag31 can bind and effectively tether Cardiolipin (CL)-containing liposomes. We further show that the tethering activity lies in the DivIVA-domain containing N-terminal of Wag31 while the C-terminal mediates protein-protein interactions of Wag31. Despite retaining its ability to interact with partner proteins, the DivIVA domain-deleted Wag31 mutant shows defects in liposome tethering in vitro and non-polar localization of CL in vivo, which eventually causes lethality. Our study suggests that membrane tethering licenses Wag31 to form scaffolds that help orchestrate protein-lipid and protein-protein interactions necessary for mycobacterial growth and survival.

microbiology↗

bioRxiv2023

Dynamics of membrane tubulation coupled with fission by a two-component module revealed on polymer cushioned bilayer islands

Membrane tubulation coupled with fission (MTCF) is a widespread cellular phenomenon but mechanisms for their coordination remain unclear. This is partly because of the lack of assays to monitor dynamics of membrane tubulation. Using polymer cushioned bilayer islands, we analyze functions of the membrane tubulator Bridging Integrator 1 (BIN1) mixed with the fission catalyst dynamin2 (Dyn2). Our results reveal that this mixture constitutes a minimal two-component module that demonstrates MTCF. MTCF is an emergent property and arises because BIN1 dually functions in facilitating peripheral recruitment while inhibiting membrane binding of Dyn2 in a dose-dependent manner. MTCF is therefore apparent only at high Dyn2 to BIN1 ratios. Because of their mutual involvement in T-tubules biogenesis, mutations in BIN1 and Dyn2 are associated with centronuclear myopathies (CNM) and our analysis links the pathology with aberrant MTCF. Together, our results establish cushioned bilayer islands as a facile template for the quantitative analysis of membrane tubulation and inform of mechanisms that coordinate MTCF.

biochemistry↗

bioRxiv2022

Mechanistic analysis of a novel membrane-interacting variable loop in the pleckstrin-homology domain critical for dynamin function

Classical dynamins are best understood for their ability to generate vesicles by membrane fission. During clathrin-mediated endocytosis (CME), dynamin is recruited to the membrane through multivalent protein and lipid interactions between its Proline Rich Domain (PRD) with SRC Homology 3 (SH3) domains in endocytic proteins and its Pleckstrin Homology Domain (PHD) with membrane lipids. Variable loops (VL) in the PHD bind lipids and partially insert into the membrane thereby anchoring the PHD to the membrane. Recent molecular dynamics (MD) simulations reveal a novel VL4 that interacts with the membrane. Importantly, a missense mutation that reduces VL4 hydrophobicity is linked to an autosomal dominant form of Charcot Marie Tooth (CMT) neuropathy. To mechanistically link data from simulations with the CMT neuropathy, we analyzed functions of a VL4 mutant with reduced hydrophobicity. In assays that rely solely on lipid-based membrane recruitment, this mutant showed an acute membrane curvature-dependent binding and fission. Remarkably, in assays that mimic a physiological multivalent lipid- and protein-based recruitment, this mutant was completely defective in fission across a range of membrane curvatures. Importantly, expression of this mutant in cells inhibited CME, consistent with the autosomal dominant phenotype associated with the CMT neuropathy. Together, our results emphasize the significance of finely tuned lipid and protein interactions for efficient dynamin function.

biochemistry↗

bioRxiv2022

Metal-binding propensity in the mitochondrial dynamin-related protein 1

Dynamin-related protein1 (Drp1) functions to divide mitochondria and peroxisomes by binding specific adaptor proteins and lipids, both of which are integral to the limiting organellar membrane. In efforts to understand how such multivalent interactions regulate Drp1 functions, in vitro reconstitution schemes rely on recruiting soluble portions of the adaptors appended with genetically encoded polyhistidine tags onto membranes containing Ni2+-bound chelator lipids. These strategies are facile and circumvent the challenge in working with membrane proteins but assume that binding is specific to proteins carrying the polyhistidine tag. Here, we find using chelator lipids and chelator beads that both native and recombinant Drp1 directly bind Ni2+ ions. Metal-binding therefore represents a potential strategy to deplete or purify Drp1 from native tissue lysates. Importantly, high concentrations of the metal in solution inhibit GTP hydrolysis and renders Drp1 inactive in membrane fission. Together, our results emphasize a metal-binding propensity, which could significantly impact Drp1 functions.

biochemistry↗

bioRxiv2021

Protein-protein interactions on membrane surfaces analysed using SUPER template pull-downs

Discovery-based proteomics workflows that identify novel interactors rely on immunoprecipitations or pull-downs with genetically-tagged bait proteins immobilized on appropriate matrices. But strategies to analyse protein interactions on a diffusible membrane surface combined with the practical ease of pull-downs remain unavailable. Such strategies are important to analyse protein complexes that mature in composition and stability because of diffusion-based encounter between participant proteins. Here, we describe a generic pull-down strategy to analyse such complexes using chelating lipid-containing supported bilayers formed on silica beads. These templates can display desired His-tagged bait proteins on a diffusible membrane surface. Using clathrin-mediated endocytosis as a paradigm, we find that the clathrin-binding adaptor protein epsin1 displayed as bait on these templates pulls down significantly higher amounts of clathrin from brain lysates than when immobilized on conventional matrices. Together, our results establish the potential of such templates as superior matrices for analysing protein-protein interactions and resultant complexes formed on membrane surfaces.

biochemistry↗

bioRxiv2019

Flexible pivoting of the pleckstrin domain domain in dynamin catalyzes fission by affecting membrane properties: Insights into the molecular degrees of freedom

The neuronal dynamin1 functions in the release of synaptic vesicles by orchestrating the process of GTPase-dependent membrane fission. Dynamin1 associates with the plasma membrane-localized phosphatidylinositol-4,5-bisphosphate (PIP2) with its centrally-located pleckstrin homology domain (PHD). The PHD is dispensable as fission can be managed, albeit at much slower rates, even when the PHD-PIP2 interaction is replaced by a generic polyhistidine- or polylysine-lipid interaction. However, even when the PHD is present, the length of the dynamin scaffold and in turn the membrane remodeling and fission rates are severely restricted with mutations such as I533A on membrane-interacting variable loop 1 (VL1) of PHD. These observations suggest that PIP2-containing membrane interactions of PHD could have evolved to expedite fission to fulfill the requirement of rapid kinetics of synaptic vesicle recycling. Here, we use a suite of multiscale modeling approaches that combine atomistic molecular dynamics simulations, mixed resolution membrane mimetic models, coarse-grained molecular simulations and advanced free-energy sampling methods (metadynamics and umbrella sampling) to explore PHD-membrane interactions. Our results reveal that: (a) the binding of PHD to PIP2-containing membranes modulates the lipids towards fission-favoring conformations and softens the membrane, (b) that PHD engages another loop (VL4) for membrane association, which acts as an auxiliary pivot and modulates the orientation flexibility of PHD on the membrane - a mechanism we believe may be important for high fidelity dynamin collar assembly on the membrane. (c) Through analyses of our trajectories data and free-energy calculations on membrane-bound WT and mutant systems, we also identify key residues on multiple VLs that stabilizes PHD membrane association. And we suggest experiments to explore the ability of PHD to associate with membrane in orientations that favors faster fission. Together, these insights provide a molecular-level understanding of the "catalytic" role of the PHD in dynamin-mediated membrane fission. SIGNIFICANCEDynamin, a large multi-domain GTPase, remodels the membrane by self-assembling onto the neck of a budding vesicle and induces fission by its energy driven conformational changes. In this work, we use multi-scale molecular simulations to probe the role of dynamins pleckstrin-homology domain (PHD), which facilitates membrane interactions. Notably, PHD is dispensable for fission as is the case with extant bacterial and mitochondrial dynamins. However, reconstitution experiments suggest that the functional role of PHD in neuronal-membrane goes beyond that of an adaptor domain as it possibly expedites the fission reaction during synaptic vesicle recycling. We provide a molecular-dynamics picture of how PHDs make membranes more pliable for fission and suggest new insights into the molecular-level processes driving the expedited fission behavior.

biophysics↗