Biology subjects

Karatekin, E.

Publications and source records attributed to Karatekin, E..

14 recordsLinked to original sources

bioRxiv2026

Type IV Secretion System Drives Lipid Mixing

Type IV secretion systems (T4SSs) are versatile molecular machines used by bacteria to secrete protein effectors into host cells, promoting pathogenesis, and to transfer DNA between bacteria through conjugation, driving horizontal gene transfer. Most, like Dot/Icm of the pathogen Legionella pneumophila (L. pneumophila) or Escherichia coli (E. coli) RK2, are primed for substrate delivery only upon contact with a target membrane, but mechanisms are unknown. A pilus could bind a receptor to initiate priming, but many T4SSs, especially those that deliver effectors, lack a pilus. Here, we present evidence that T4SSs are primed by direct contact with target membrane lipids. Combining fluorescence assays with genetics and biochemistry, we found that Dot/Icm drives lipid exchange between bacterial cells and between bacteria and synthetic membranes containing only lipids. Lipid exchange requires membrane contact but does not require ATP hydrolysis or even full complex assembly. Minimally, the outer membrane core complex protein DotG needs to be present in at least one of the apposed membranes. We similarly observed lipid mixing with the simpler E. coli RK2 T4SS, where we could follow lipid mixing and plasmid transfer simultaneously. We found that lipid mixing always preceded or accompanied plasmid transfer, suggesting it may be part of the contact-dependent priming mechanism. Lipid mixing was inhibited or promoted by lipids that inhibit or promote membrane fusion, respectively. Lipids inhibiting lipid mixing also inhibited substrate transfer. Together, our results suggest that initial contact between DotG outer segments and target membrane lipids promotes lipid mixing as part of the mechanism that primes T4SS for substrate translocation. HighlightsO_LIThe Legionella pneumophila Dot/Icm secretion system requires contact with a target membrane for effector translocation, but how membrane contact primes the machinery for this is unknown. C_LIO_LIWe found that Dot/Icm drives contact-dependent lipid mixing between bacteria or between bacteria and liposomes, showing that Dot/Icm priming does not require a protein receptor. C_LIO_LILipid mixing also occurs with the simpler Type IVA system (Escherichia coli RK2) during conjugation, suggesting lipid mixing is a general feature of Type IV Secretion System function. C_LIO_LIThe core complex protein DotG is sufficient to drive lipid mixing, suggesting DotG-target membrane interactions may destabilize the target membrane. C_LI

microbiology↗

bioRxiv2025

Electrophysiological profiling of exocytosis during early-stage development of the zebrafish lateral line

Hair cells of the zebrafish lateral line have proven to be a good model for studying hair cell function in a system that is easily genetically manipulated, rapidly develops and is experimentally accessible. However, characterization of potential developmental changes, and possible differences along lateral line position are lacking. Here, we used in vivo patch clamp to investigate the electrophysiological and exocytic properties of neuromast hair cells over early development across body location. Long depolarizations led to steady increases in membrane capacitance, presumably due to exocytosis of vesicles localized to ribbon synapses. The magnitude and kinetics of capacitance changes did not vary significantly across the L1 to L6 position of neuromasts along lateral line, but the magnitudes were found to be significantly smaller in hair cells found in the tail region across all developmental time points. For each region, we found no significant changes in capacitance responses between 3 and 7 days after fertilization. Hair cell capacitance responses were greatly reduced in animals injected with CRISPR/Cas9 with gRNAs targeted to otoferlin b. These results confirm the essential role of otoferlin b in neuromast hair cell function, and they establish the fidelity of CRISPR/Cas9 to rapidly mediate genetic removal of critical genes to study their impact on synaptic release.

neuroscience↗

bioRxiv2024

Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1

Synaptotagmin-1 (Syt1) is a major calcium sensor for rapid neurotransmitter release in neurons and hormone release in many neuroendocrine cells. It possesses two tandem cytosolic C2 domains that bind calcium, negatively charged phospholipids, and the neuronal SNARE complex. Calcium binding to Syt1 triggers exocytosis, but how this occurs is not well understood. Syt1 has additional roles in docking dense core vesicles (DCV) and synaptic vesicles (SV) to the plasma membrane (PM) and in regulating fusion pore dynamics. Thus, Syt1 perturbations could affect release through vesicle docking, fusion triggering, fusion pore regulation, or a combination of these. Here, using a human neuroendocrine cell line, we show that neutralization of highly conserved polybasic patches in either C2 domain of Syt1 impairs both DCV docking and efficient release of serotonin from DCVs. Interestingly, the same mutations resulted in larger fusion pores and faster release of serotonin during individual fusion events. Thus, Syt1s roles in vesicle docking, fusion triggering, and fusion pore control may be functionally related.

biophysics↗

bioRxiv2024

Cellular function of the GndA small open reading frame-encoded polypeptide during heat shock

Hundreds of bacterial small open reading frames (sORFs) encoding microproteins of fewer than fifty amino acids have recently been identified. Biological functions have been ascribed to an increasing number of microproteins, some of which play roles in bacterial stress responses. In this work, we provide evidence that GndA, a frameshifted 36-amino acid microprotein encoded in an internal open reading frame overlapping the 6-phosphogluconate dehydrogenase (6PGD) coding sequence (CDS), functions in the response to heat shock. We demonstrate independent contributions of GndA and 6PGD to cell growth at high temperature. GndA is hydrophobic and associates with the pyruvate dehydrogenase complex and respiratory complex I (RCI). Consistent with a role in cellular homeostasis, GndA mutations alter the transcriptional responses to heat shock. These results demonstrate that GndA promotes bacterial cell growth at high temperature via a mechanism distinct from that of the 6PGD protein that it overlaps. GndA thus expands the paradigm of bacterial microproteins that function in cellular stress responses.

biochemistry↗

bioRxiv2021

Membrane fission during bacterial spore development requires DNA-driven cellular inflation

Bacteria require membrane fission for cell division and endospore formation. FisB catalyzes membrane fission during sporulation, but the molecular basis is unclear as it cannot remodel membranes by itself. Sporulation initiates with an asymmetric division that generates a large mother cell and a smaller forespore that contains only 1/4 of its complete genome. As the mother cell membranes engulf the forespore, a DNA translocase pumps the rest of the chromosome into the small forespore compartment, inflating it due to increased turgor. When the engulfing membranes undergo fission, the forespore is released into the mother cell cytoplasm. Here we show that forespore inflation and FisB accumulation are both required for efficient membrane fission. We suggest that high membrane tension in the engulfment membrane caused by forespore inflation drives FisB-catalyzed membrane fission. Collectively our data indicate that DNA-translocation has a previously unappreciated second function in energizing FisB-mediated membrane fission under energy-limited conditions. HIGHLIGHTS- Membrane fission during endospore formation requires rapid forespore inflation by ATP-driven DNA translocation. - Forespore inflation is fast enough to increase the tension of the engulfment and forespore membranes to near lysis tensions. - FisB catalyzes membrane fission by impeding the flux of lipids that partially support forespore and engulfment membrane growth. - Membrane fission utilizes chemical energy transduced to mechanical energy during DNA-packing into the forespore.

biophysics↗

bioRxiv2021

Polybasic patches in both C2 domains of Synaptotagmin-1 are required for evoked neurotransmitter release

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release. It is composed of a single-pass transmembrane domain linked to two tandem C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is not well understood. Calcium binding to C2B, but not to C2A, is critical for synchronous release and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here we asked what critical feature of C2A may be responsible for its functional role, and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side-by-side to determine their relative contributions to Syt1 function in cultured cortical mouse neurons and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The readily releasable vesicle pool or spontaneous release were not affected, so both patches are specifically required for synchronization of release. We suggest these patches contribute to cooperative binding to membranes, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release. Significance StatementSynaptotagmin-1 is a vesicular calcium sensor required for synchronous neurotransmitter release. Its tandem cytosolic C2 domains (C2A and C2B) bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. How calcium-binding to Synaptotagmin-1 leads to release and the relative contributions of the two C2 domains is not clear: unlike C2B, calcium-binding to C2A is not critical for evoked release, yet both domains are needed for Syt1 function. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements that probe single C2 domain-membrane interactions, we show that conserved polybasic regions in both domains contribute to membrane binding cooperatively, and both are required for evoked release, likely by increasing the overall affinity of Synaptotagmin-1 for negatively charged membranes.

neuroscience↗

bioRxiv2021

Rapid propagation of membrane tension at a presynaptic terminal

Many cellular activities, such as cell migration1, cell division2,3, signaling4,5, infection6, phagocytosis7 and exo-endocytosis8-11, generate membrane tension gradients that in turn regulate them12. Moreover, membrane flows, which are driven by tension gradients, can limit exo-endocytosis coupling in space and time, as net membrane flow from exocytic to endocytic sites is required to maintain membrane homeostasis13. However, there is controversy over how rapidly plasma membrane flows can relax tension gradients; contrary to the common view12,14,15, recent work showed membrane tension does not equilibrate in several cell types16. Here we show membrane tension can propagate rapidly or slowly, spanning orders of magnitude in speed, depending on cell type. In a neuronal terminal specialized for rapid synaptic vesicle turnover and where exo-endocytosis events occur at distinct loci, membrane tension equilibrates within seconds. By contrast, membrane tension does not propagate in neuroendocrine adrenal chromaffin cells secreting catecholamines. Thus, slow membrane flow and tension equilibration may confine exo- and exocytosis to the same loci17. Stimulation of exocytosis causes a rapid, global decrease in the synaptic terminal membrane tension, which recovers slowly due to endocytosis. Our results demonstrate membrane tension propagates rapidly at neuronal terminals and varies during synaptic activity, likely contributing to exo-endocytosis coupling.

biophysics↗

bioRxiv2021

Myosins generate contractile force and maintain organization in the cytokinetic contractile ring

During cytokinesis cells assemble an actomyosin contractile ring whose tension constricts and divides cells, but the ring tension was rarely measured. Actomyosin force generation is well understood for the regular sarcomeric architecture of striated muscle, but recent super-resolution studies of fission yeast contractile rings revealed organizational building blocks that are not sarcomeres but irregularly positioned plasma membrane-anchored protein complexes called nodes. Here we measured contractile ring tensions in fission yeast protoplast cells. The myosin II isoforms Myo2 and Myp2 generated the tension, with a ~2-fold greater contribution from Myo2. Simulations of a molecularly detailed ring model revealed a sliding node mechanism for tension, where nodes hosting tense actin filaments were pulled bidirectionally around the ring. Myo2 and Myp2 chaperoned self-assembling components into the ring organization, and anchored the ring against bridging instabilities. Thus, beyond force production, Myo2 and Myp2 are the principal organizers, bundlers and anchors of the contractile ring.

cell biology↗

bioRxiv2020

Requirements for FisB-mediated membrane fission during sporulation

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, FisB, is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of [~]12 molecules that give way to an immobile cluster at the engulfment pole containing [~]40 proteins at the time of membrane fission. Analysis of FisB mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Experiments using artificial membranes and filamentous cells suggest FisB does not have an intrinsic ability to sense or induce membrane curvature but can bridge membranes. Finally, modeling suggests homo-oligomerization and trans interactions with membranes are sufficient to explain FisB accumulation at the membrane neck that connects the engulfment membrane to the rest of the mother cell membrane during late stages of engulfment. Together, our results show that FisB is a robust and unusual membrane fission protein that relies on homo-oligomerization, lipid-binding and the unique membrane topology generated during engulfment for localization and membrane scission, but surprisingly, not on lipid microdomains, negative-curvature lipids, or curvature-sensing.

biophysics↗

bioRxiv2020

DNA-Origami-Based Fluorescence Brightness Standards for Convenient and Fast Protein Counting in Live Cells

Fluorescence microscopy has been one of the most discovery-rich methods in biology. In the digital age, the discipline is becoming increasingly quantitative. Virtually all biological laboratories have access to fluorescence microscopes, but abilities to quantify biomolecule copy numbers are limited by the complexity and sophistication associated with current quantification methods. Here, we present DNA-origami-based fluorescence brightness standards for counting 5-300 copies of proteins in mammalian and bacterial cells, tagged with fluorescent proteins or organic dyes. Compared to conventional quantification techniques, our brightness standards are robust, straightforward to use, and compatible with nearly all fluorescence imaging applications, thereby providing a practical and versatile tool to quantify biomolecules via fluorescence microscopy.

biophysics↗

bioRxiv2020

Retromer forms low order oligomers on supported lipid bilayers

Retromer is a protein sorting device that orchestrates the selection and export of integral membrane proteins from the endosome via retrograde and plasma membrane recycling pathways. Long standing hypotheses regarding the Retromer sorting mechanism posit that oligomeric interactions between Retromer and associated accessory factors on the endosome membrane drives clustering of Retromer-bound integral membrane cargo prior to its packaging into a nascent transport carrier. To test this hypothesis, we examined interactions between the components of the SNX3-Retromer sorting pathway using quantitative single particle fluorescence microscopy of a reconstituted system comprising a supported bilayer, Retromer, a model cargo protein, the accessory proteins SNX3, RAB7, and the Retromer-binding segment of the WASHC2C subunit of the WASH complex. The predominant species of membrane associated Retromer are low order: monomers ([~]18%), dimers ([~]35%), trimers ([~]24%) and tetramers ([~]24%). Unexpectedly, neither cargo nor accessory factors promote Retromer oligomerization on a supported bilayer. The results indicate that Retromer has an intrinsic propensity to form low order oligomers and that neither membrane association nor accessory factors potentiate oligomerization. Hence, Retromer is a minimally concentrative sorting device adapted to bulk membrane trafficking from the endosomal system.

cell biology↗

bioRxiv2020

Sorting liposomes of distinct sizes by DNA-brick assisted centrifugation

The "tiny bubbles of fluid" wrapped by lipid-bilayer membranes, termed vesicles, are abundant in cells and extracellular space, performing critical tasks including nutrient uptake, cargo transport, and waste confinement. Vesicles on different missions and transport routes are often distinct both in size and in chemical composition, which confers specificity to their interactions with other membranous compartments. Therefore, to accurately recapitulate the vesicles structure and behavior, it is important to use homogeneous liposomes (vesicles made of synthetic components) with precisely defined attributes as model membranes. Although existing methods can generate liposomes of selected sizes with reasonable homogeneity, the scalable production of uniformly-sized liposomes across a wide range of dimensions and compositions remains challenging. Here we report a streamlined, high-throughput sorting technique that uses cholesterol-modified "nanobricks" made of a few DNA oligonucleotides to differentiate hetero-sized liposomes by their buoyant densities. After DNA-brick coating, milligrams of liposomes of different origins (e.g., produced via extrusion or sonication, and reconstituted with membrane proteins) can be separated by centrifugation into six to eight homogeneous populations with mean diameters from 30 to 130 nm. In proof of concept experiments, we show that these uniform, leak-free liposomes are ideal substrates to study, with an unprecedented resolution, how membrane curvature influences the activity of peripheral (ATG3) and integral (SNARE) membrane proteins. We anticipate that our sorting technique will facilitate the quantitative understanding of membrane curvature in vesicular transport. Furthermore, adding a facile and standardized separation step to the conventional liposome preparation pipeline may benefit the formulation and prototyping of liposomal drug-carrying vehicles.

biophysics↗

bioRxiv2019

The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores mechanically

All membrane fusion reactions proceed through an initial fusion pore, including calcium-triggered release of neurotransmitters and hormones. Expansion of this small pore to release cargo is energetically costly and regulated by cells, but the mechanisms are poorly understood. Here we show that the neuronal/exocytic calcium sensor Synaptotagmin-1 (Syt1) promotes expansion of fusion pores induced by SNARE proteins. Pore dilation relied on calcium-induced insertion of the tandem C2 domain hydrophobic loops of Syt1 into the membrane, previously shown to reorient the C2 domain. Mathematical modelling suggests that C2B reorientation rotates a bound SNARE complex so that it exerts force on the membranes in a mechanical lever action that increases the height of the fusion pore, provoking pore dilation to offset the bending energy penalty. We conclude that Syt1 exerts novel non-local calcium-dependent mechanical forces on fusion pores that dilate pores and assist neurotransmitter and hormone release. SIGNIFICANCE STATEMENTDuring neurotransmitter release, calcium-induced membrane insertion of the C2B domain of Synaptotagmin re-orients the bound SNARE complex which dilates the fusion pore in a mechanical lever action.

biophysics↗

bioRxiv2017

Single-molecule force spectroscopy of protein-membrane interactions

Many biological processes rely on protein-membrane interactions in the presence of mechanical forces, yet high resolution methods to quantify such interactions are lacking. Here, we describe a single-molecule force spectroscopy approach to quantify membrane binding of C2 domains in Synaptotagmin-1 (Syt1) and Extended Synaptotagmin-2 (E-Syt2). Syts and E-Syts bind the plasma membrane via multiple C2 domains, bridging the plasma membrane with synaptic vesicles or endoplasmic reticulum to regulate membrane fusion or lipid exchange, respectively. In our approach single proteins attached to membranes supported on silica beads are pulled by optical tweezers, allowing membrane binding and unbinding transitions to be measured with unprecedented spatiotemporal resolution. C2 domains from either protein resisted unbinding forces of 2-7 pN and had binding energies of 4-14 kBT per C2 domain. Regulation by bilayer composition or Ca2+ recapitulated known properties of both proteins. The method can be widely applied to study protein-membrane interactions.

biophysics↗