Samsuzzoha Mondal

All Publications

• Endophilin-lamellipodin-VASP, key components in fast endophilin-mediated endocytosis, control actin polymerization within liquid-like condensates. The Journal of biological chemistry Narayan, K. B., James, H. P., Cope, J., Mondal, S., Baeyens, L., Milano, F., Zheng, J., Krause, M., Baumgart, T. 2025; 301 (12): 110834

  Abstract

  Actin polymerization is essential in several clathrin-independent endocytic pathways including fast endophilin-mediated endocytosis (FEME); however, the actin machinery involved in FEME has been elusive. Here, we show that the actin polymerase VASP colocalizes and interacts directly with the FEME priming complex. We identify endophilin as a VASP binding partner and establish novel noncanonical interactions between the SH3 domain of endophilin and the Ena/VASP homology 1 or 2 (EVH1 and EVH2) domains of VASP. We show that liquid-like condensates formed by endophilin, lamellipodin, and VASP localize actin polymerization both in solution and on lipid membranes and result in the formation of torus-shaped condensates in the bulk. We establish a new role for multivalent endophilin-lamellipodin interactions in the regulation of actin polymerization, and we identify a novel function for endophilin as a promoter of actin bundling. Our findings support a model that explains the connection between local actin polymerization and dynamic formation and dissolution of endocytic priming patches in FEME.

  View details for DOI 10.1016/j.jbc.2025.110834

  View details for PubMedID 41109347

  View details for PubMedCentralID PMC12663515

• Detection of PIP2 distributions in biological membranes using a peptide-based sensor. The Journal of biological chemistry Menon, V. K., Wu, J., Alonzo, A. J., Scrudders, K. L., Rogers, K. A., Selvarajan, S., Walke, A., Pellicier-Caraballo, J., Kundu, R., Mondal, S., Datta, A., Low-Nam, S. T. 2025: 110826

  Abstract

  Organization and composition of the plasma membrane are important modulators of many cellular programs. Phosphatidylinositol phosphate (PIP) lipids are low abundance membrane constituents with different arrangements of phosphate groups around an inositol head group and that regulate many signaling pathways. Numerous strategies have been developed to detect and track PIP species to monitor their clustering, mobility, and interactions with binding partners. We implement a peptide-based, ratiometric sensor for the detection of PI(4,5)P2 lipids in reconstituted membrane systems that permit absolute quantification of PI(4,5)P2 densities down to physiological levels less than four mole percent. The sensor is membrane permeable and easily transferable to measurements in living cells. Application of calibrated sensors to cells expressing common mutations in the small GTPase, Ras, showed a reshaping of surface PI(4,5)P2 levels and distributions in a mutation-specific manner. Brief treatment of G12C mutant Ras cells with the specific inhibitor, Sotorasib, resulted in alterations to surface PI(4,5)P2 arrangements that resemble the wild-type (WT) Ras. Thus, the rapid redistribution of PI(4,5)P2 lipids upon drug treatment emphasizes the tight coupling between membrane composition, organization, and downstream signaling outcomes. Tools and strategies to monitor membrane composition alongside cellular behaviors could provide pipelines to characterize therapeutics and improve the mechanistic understanding of how protein-lipid coupling drives cellular programs.

  View details for DOI 10.1016/j.jbc.2025.110826

  View details for PubMedID 41109350

• A cell-permeable fluorescent probe reveals temporally diverse PI(4,5)P2 dynamics evoked by distinct GPCR agonists in neurons. Chemical science Kundu, R., Mondal, S., Kapadia, A., Banerjee, A. A., Kucherak, O. A., Klymchenko, A. S., Koushika, S. P., Venkatramani, R., Vaidya, V. A., Datta, A. 2025; 16 (24): 10970-10982

  Abstract

  Lipids, key constituents of cell-membranes, are the first responders to cell signals. At the crux of spatiotemporal dynamics of lipid-signaling responses are phosphoinositides. Indeed, phosphoinositides like phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2), present in the inner-leaflet of eukaryotic cell-membranes, form the link between signal reception and downstream signal-transmission. In this backdrop, reversible fluorescent probes that can track live PI(4,5)P2 dynamics on a seconds time-scale will afford key insights into lipid-mediated signaling. However, realizing cell-permeable PI(4,5)P2-selective sensors for imaging dynamics remains a challenge due to the presence of structurally similar lipids and low levels of PI(4,5)P2. We report a computationally-designed, rapid-response, reversible, photo-stable, fluorescent sensor that permeates living cells, neurons, and a multicellular organism within few min of direct incubation and distinctly visualizes PI(4,5)P2 pools. We used the sensor to interrogate the role of PI(4,5)P2 in driving the heterogeneity of signaling responses and contrasting behavioral effects that ensue upon binding of distinct ligands to the same G protein-coupled receptor. Specifically, we asked whether probing PI(4,5)P2 dynamics using our novel sensor could uncover the earliest of signaling differences evoked by hallucinogenic versus non-hallucinogenic ligands at the serotonin2A (5-HT2A) receptor. Our results reveal that a hallucinogenic ligand at the 5-HT2A receptor leads to a slower rate of PI(4,5)P2-depletion when compared to a non-hallucinogenic ligand, within the initial seconds of ligand addition, but has a sustained effect. The ability of our designer chemical probe in timing early seconds-minute timescale lipid-dynamics in living cells opens avenues for tracking early time-point molecular events in neuronal response to chemical and physical stimuli.

  View details for DOI 10.1039/d5sc01306b

  View details for PubMedID 40401190

  View details for PubMedCentralID PMC12090097

• Magnetic resonance control of reaction yields through genetically-encoded protein:flavin spin-correlated radicals in a live animal. bioRxiv : the preprint server for biology Burd, S. C., Bagheri, N., Ingaramo, M., Condon, A. F., Mondal, S., Dowlatshahi, D. P., Summers, J. A., Mukherjee, S., York, A. G., Wakatsuki, S., Boxer, S. G., Kasevich, M. 2025

  Abstract

  Radio-frequency (RF) magnetic fields can influence reactions involving spin-correlated radical pairs. This provides a mechanism by which RF fields can influence living systems at the biomolecular level. Here we report the modification of the emission of various red fluorescent proteins (RFPs), in the presence of a flavin cofactor, induced by a combination of static and RF magnetic fields. Resonance features in the protein fluorescence intensity were observed near the electron spin resonance frequency at the corresponding static magnetic field strength. This effect was measured at room temperature both in vitro and in the nematode C. elegans , genetically modified to express the RFP mScarlet. These observations suggest that the magnetic field effects measured in RFP-flavin systems are due to quantum-correlated radical pairs. Our experiments demonstrate that RF magnetic fields can influence dynamics of reactions involving RFPs in biologically relevant conditions, and even within a living animal. These results have implications for the development of a new class of genetic tools based on RF manipulation of genetically-encoded quantum systems.

  View details for DOI 10.1101/2025.02.27.640669

  View details for PubMedID 40093161

  View details for PubMedCentralID PMC11908193

• Purification of Recombinant Human Amphiphysin 1 and its N-BAR Domain. Bio-protocol Mondal, S., James, H. P., Milano, F., Jin, R., Baumgart, T. 2023; 13 (12): e4699

  Abstract

  Bin/Amphiphysin/Rvs (BAR) proteins are known as classical membrane curvature generators during endocytosis. Amphiphysin, a member of the N-BAR sub-family of proteins that contain a characteristic amphipathic sequence at the N-terminus of the BAR domain, is involved in clathrin-mediated endocytosis. Full-length amphiphysin contains a ~ 400 amino acid long disordered linker connecting the N-BAR domain and a C-terminal Src homology 3 (SH3) domain. We express and purify recombinant amphiphysin and its N-BAR domain along with an N-terminal glutathione-S-transferase (GST) tag. The GST tag allows extraction of the protein of interest using affinity chromatography and is removed in the subsequent protease treatment and ion-exchange chromatography steps. In the case of the N-BAR domain, cleavage of the GST tag was found to cause precipitation. This issue can be minimized by adding glycerol to the protein purification buffers. In the final step, size exclusion chromatography removes any potential oligomeric species. This protocol has also been successfully used to purify other N-BAR proteins, such as endophilin, Bin1, and their corresponding BAR domains. Graphical overview.

  View details for DOI 10.21769/BioProtoc.4699

  View details for PubMedID 37397795

  View details for PubMedCentralID PMC10308189

• Membrane reshaping by protein condensates. Biochimica et biophysica acta. Biomembranes Mondal, S., Baumgart, T. 2023; 1865 (3): 184121

  Abstract

  Proteins can organize into dynamic, functionally important assemblies on fluid membrane surfaces. Phase separation has emerged as an important mechanism for forming such protein assemblies on the membrane during cell signaling, endocytosis, and cytoskeleton regulation. Protein-protein phase separation thus adds novel fluid mosaics to the classical Singer and Nicolson model. Protein condensates formed in this process can modulate membrane morphologies. This is evident from recent reports of protein condensate-driven membrane reshaping in processes such as endocytosis, autophagosome formation, and protein storage vacuole morphogenesis in plants. Lateral phase separation (on the membrane surface) of peripheral curvature coupling proteins can modulate such membrane morphological transitions. Additionally, three-dimensional protein phase separation can result in droplets that through adhesion can affect membrane shape changes. How do these condensate-driven curvature generation mechanisms contrast with the classically recognized scaffolding and amphipathic helix insertion activities of specific membrane remodeling proteins? A salient feature of these condensate-driven membrane activities is that they depend upon both macroscopic features (such as interfacial energies of the condensate, membrane, and cytosol) as well as microscopic, molecular-level interactions (such as protein-lipid binding). This review highlights the current understanding of the mechanisms underlying curvature generation by protein condensates in various biological pathways.

  View details for DOI 10.1016/j.bbamem.2023.184121

  View details for PubMedID 36642341

  View details for PubMedCentralID PMC10208392

• Complex multivalent protein interactions involving endophilin, lamellipodin and VASP regulate membrane shaping phenomena and actin assembly Narayan, K. B., Mondal, S., James, H., Baeyens, L., Zheng, J., Baumgart, T. CELL PRESS. 2023: 483A

  View details for Web of Science ID 000989629702598

• Phase separation can regulate membrane curvature generation by bar-proteins Mondal, S., Narayan, K., Baumgart, T. CELL PRESS. 2023: 30A

  View details for Web of Science ID 000989629700147

• Multivalent interactions between molecular components involved in fast endophilin mediated endocytosis drive protein phase separation. Nature communications Mondal, S., Narayan, K., Botterbusch, S., Powers, I., Zheng, J., James, H. P., Jin, R., Baumgart, T. 2022; 13 (1): 5017

  Abstract

  A specific group of transmembrane receptors, including the β1-adrenergic receptor (β1-AR), is internalized through a non-clathrin pathway known as Fast Endophilin Mediated Endocytosis (FEME). A key question is: how does the endocytic machinery assemble and how is it modulated by activated receptors during FEME. Here we show that endophilin, a major regulator of FEME, undergoes a phase transition into liquid-like condensates, which facilitates the formation of multi-protein assemblies by enabling the phase partitioning of endophilin binding proteins. The phase transition can be triggered by specific multivalent binding partners of endophilin in the FEME pathway such as the third intracellular loop (TIL) of the β1-AR, and the C-terminal domain of lamellipodin (LPD). Other endocytic accessory proteins can either partition into, or target interfacial regions of, these condensate droplets, and LPD also phase separates with the actin polymerase VASP. On the membrane, TIL promotes protein clustering in the presence of endophilin and LPD C-terminal domain. Our results demonstrate how the multivalent interactions between endophilin, LPD, and TIL regulate protein assembly formation on the membrane, providing mechanistic insights into the priming and initiation steps of FEME.

  View details for DOI 10.1038/s41467-022-32529-0

  View details for PubMedID 36028485

  View details for PubMedCentralID PMC9418313

• Endophilin recruitment drives membrane curvature generation through coincidence detection of GPCR loop interactions and negative lipid charge. The Journal of biological chemistry Mondal, S., Narayan, K. B., Powers, I., Botterbusch, S., Baumgart, T. 2021; 296: 100140

  Abstract

  Endophilin plays key roles during endocytosis of cellular receptors, including generating membrane curvature to drive internalization. Electrostatic interactions between endophilin's BIN/Amphiphysin/Rvs domain and anionic membrane lipids have been considered the major driving force in curvature generation. However, the SH3 domain of endophilin also interacts with the proline-rich third intracellular loop (TIL) of various G-protein-coupled receptors (GPCRs), and it is unclear whether this interaction has a direct role in generating membrane curvature during endocytosis. To examine this, we designed model membranes with a membrane density of 1400 receptors per μm2 represented by a covalently conjugated TIL region from the β1-adrenergic receptor. We observed that TIL recruits endophilin to membranes composed of 95 mol% of zwitterionic lipids via the SH3 domain. More importantly, endophilin recruited via TIL tubulates vesicles and gets sorted onto highly curved membrane tubules. These observations indicate that the cellular membrane bending and curvature sensing activities of endophilin can be facilitated through detection of the TIL of activated GPCRs in addition to binding to anionic lipids. Furthermore, we show that TIL electrostatically interacts with membranes composed of anionic lipids. Therefore, anionic lipids can modulate TIL/SH3 domain binding. Overall, our findings imply that an interplay between TIL, charged membrane lipids, BAR domain, and SH3 domain could exist in the biological system and that these components may act in coordination to regulate the internalization of cellular receptors.

  View details for DOI 10.1074/jbc.RA120.016118

  View details for PubMedID 33268381

  View details for PubMedCentralID PMC7948419

• A Bishydrated, Eight-Coordinate Gd(III) Complex with Very Fast Water Exchange: Synthesis, Characterization, and Phantom MR Imaging CHEMISTRYSELECT Phukan, B., Malikidogo, K. P., Bonnet, C. S., Toth, E., Mondal, S., Mukherjee, C. 2018; 3 (27): 7668-7673

  View details for DOI 10.1002/slct.201801629

  View details for Web of Science ID 000439756200001

• Optically sensing phospholipid induced coil-helix transitions in the phosphoinositide-binding motif of gelsolin. Faraday discussions Mondal, S., Chandra, A., Venkatramani, R., Datta, A. 2018; 207 (0): 437-458

  Abstract

  We present a systematic experimental and computational study of phospholipid induced peptide coil-helix transitions which are relevant in the context of proteins mediating cytoskeletal rearrangement via membrane binding. We developed a sensitive Förster resonance energy transfer (FRET) based assay to address whether coil-helix transitions in phospholipid binding motifs of actin-binding proteins can be induced by physiologically-relevant concentrations (1-20 μM) of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) phospholipids. Based on inter-residue distance constraints obtained from Molecular Dynamics (MD) simulations of a 20 residue peptide (Gel 150-169) from the actin-severing protein gelsolin, we synthetized and labeled the peptide with a tryptophan donor and IAEDANS acceptor pair. Upon addition of PI(4,5)P2 micelles and mixed vesicles containing PI(4,5)P2 and phosphatidylcholine to the peptide, we observed a decrease in the tryptophan emission intensity with increasing concentrations of PI(4,5)P2. The IAEDANS emission spectra showed a more complex profile exhibiting a blue shift of the emission peak and non-monotonic changes in the intensity profile with increasing concentrations of PI(4,5)P2. We showed that the IAEDANS acceptor emission response is a result of both intrinsic polarity sensitivity of the acceptor in the vicinity of the membrane surface and fluorescence energy transfer from the donor. Importantly, the fluorescence lifetime of the donor (tryptophan) showed a monotonous decrease with increasing mol% of PI(4,5)P2 from 1.13 ± 0.10 ns in the absence of phospholipids to 0.25 ± 0.03 ns in the presence of 100% PI(4,5)P2 micelles. We also showed a concomitant increase in FRET efficiency with increasing PI(4,5)P2 levels indicating a PI(4,5)P2 concentration dependent coil-helix transition. Our studies demonstrate that membrane PI(4,5)P2 concentrations as low as 2.5-5 μM can trigger helix-coil conformational changes in gelsolin relevant for triggering regulatory processes in the cell.

  View details for DOI 10.1039/c7fd00197e

  View details for PubMedID 29363700

• Cell Permeable Ratiometric Fluorescent Sensors for Imaging Phosphoinositides. ACS chemical biology Mondal, S., Rakshit, A., Pal, S., Datta, A. 2016; 11 (7): 1834-43

  Abstract

  Phosphoinositides are critical cell-signal mediators present on the plasma membrane. The dynamic change of phosphoinositide concentrations on the membrane including clustering and declustering mediates signal transduction. The importance of phosphoinositides is scored by the fact that they participate in almost all cell-signaling events, and a defect in phosphoinositide metabolism is linked to multiple diseases including cancer, bipolar disorder, and type-2 diabetes. Optical sensors for visualizing phosphoinositide distribution can provide information on phosphoinositide dynamics. This exercise will ultimately afford a handle into understanding and manipulating cell-signaling processes. The major requirement in phosphoinositide sensor development is a selective, cell permeable probe that can quantify phosphoinositides. To address this requirement, we have developed short peptide-based ratiometric fluorescent sensors for imaging phosphoinositides. The sensors afford a selective response toward two crucial signaling phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol-4-phosphate (PI4P), over other anionic membrane phospholipids and soluble inositol phosphates. Dissociation constant values indicate up to 4 times higher probe affinity toward PI(4,5)P2 when compared to PI4P. Significantly, the sensors are readily cell-permeable and enter cells within 15 min of incubation as indicated by multiphoton excitation confocal microscopy. Furthermore, the sensors light up signaling phosphoinositides present both on the cell membrane and on organelle membranes near the perinuclear space, opening avenues for quantifying and monitoring phosphoinositide signaling.

  View details for DOI 10.1021/acschembio.6b00067

  View details for PubMedID 27082310

• Molecular crowding causes narrowing of population heterogeneity and restricts internal dynamics in a protein METHODS AND APPLICATIONS IN FLUORESCENCE Mondal, S., Kallianpur, M. V., Udgaonkar, J. B., Krishnamoorthy, G. 2016; 4 (1)

  View details for DOI 10.1088/2050-6120/4/1/014003

  View details for Web of Science ID 000376167600005

• Smart "lanthano" proteins for phospholipid sensing. Inorganic chemistry Gupta, S., Mondal, S., Mhamane, A., Datta, A. 2013; 52 (21): 12314-6

  Abstract

  Metal-ion-mediated interactions between calcium-binding peripheral proteins and membrane phospholipids is a key feature of multiple cell signaling processes. The molecular basis for the interaction involves the displacement of inner-sphere water molecules on calcium ions by phosphate groups of the phospholipids. On the basis of this fundamental mechanism, we have devised a novel "turn-on" optical sensing strategy for anionic phospholipids by using a lanthanide reconstituted protein. The "lanthano" protein turns on selectively in the presence of a crucial signaling phospholipid, phosphatidylserine, by affording a 6 times enhancement in lanthanide luminescence. The "turn-on" sensing strategy was distinctly validated by direct evidence for the water-displacement mechanism via lifetime measurements.

  View details for DOI 10.1021/ic4018932

  View details for PubMedID 24128273