Biology subjects

Kukulski, W.

Publications and source records attributed to Kukulski, W..

18 recordsLinked to original sources

bioRxiv2026

Adaptor interactions trigger Pan1 self-assembly during clathrin-mediated endocytosis in budding yeast

Clathrin-mediated endocytosis requires the coordinated assembly of a highly dynamic protein network that couples membrane remodeling to actin-driven force generation. The essential budding yeast protein, Pan1, scaffolds the endocytic protein network by linking adaptor proteins, and other coat components, and actin assembly regulators. How Pan1 coordinates all these different proteins in space and time is not well understood. We show that Pan1 undergoes biomolecular condensation. Elevated Pan1 expression induced the formation of condensates that exhibited partial rapid molecular exchange, temperature-dependent reversibility, and sensitivity to disruption of weak hydrophobic interactions. The Pan1 assemblies are compositionally selective, preferentially enriching late-stage endocytic factors while excluding early adaptor proteins. Truncation analysis demonstrated that condensation depends on cooperative contributions from intrinsically disordered regions, the EH2 domain, and the oligomerization module, whereas a C-terminal region negatively regulates condensation. Deletion of Pan1 regions promoting its self-assembly perturbed Pan1 assembly and function at endocytic sites. Disruption of interactions between Pan1 and endocytic adaptors did not alter the dynamics of endocytic events but instead reduced the number of endocytic events marked by Pan1 and caused the formation of ectopic Pan1 condensates. This indicates that adaptor-mediated interactions spatially constrain Pan1 localization by seeding Pan1 assembly at endocytic sites. Our findings support a model in which adaptor proteins seed Pan1 assembly at the endocytic sites while multivalent interactions drive Pan1 self-assembly to build the higher-order molecular network of the late endocytic coat.

cell biology↗

bioRxiv2025

The molecular basis of tricalbin-mediated membrane contact site organization in cells

Membrane contact sites facilitate molecular exchanges through physical interactions between organelles, connected by specific protein tethers. Among these tethers are the tricalbins, which mediate contacts between endoplasmic reticulum (ER) and plasma membrane in yeast. Tricalbins are integral to the ER, have a cytosolic lipid binding domain and bind the plasma membrane through C2 domains. Here, we combine fluorescence recovery after photobleaching with correlative light and 3D electron microscopy to dissect how tricalbins control their localization, dynamic distribution and contact site organization. We find that heteromerization via lipid binding domains is a prerequisite for tricalbin accumulation at contact sites, membrane curvature sensing and restrained mobility in the ER. By altering tricalbin protein domains, we show that intermembrane distances and intrinsically disordered regions interdependently control distribution and dynamics of contact site tethers. Our study reveals principles of contact site architecture that are fine-tuned by tricalbin domain organization.

cell biology↗

bioRxiv2025

In-Situ High-Resolution Cryo-EM Reconstructions from CEMOVIS

Cryo-electron microscopy can be used to image cells and tissue at high resolution. To ensure electron transparency, sample thickness must not exceed 500 nm. Focused-ion-beam (FIB) milling has become the standard method to prepare thin samples (lamellae), however, the material removed by the milling process is lost, the imageable area is usually limited to a few square microns, and the surface layers sustain damage from the ion beam. We have examined cryo-electron microscopy of vitreous sections (CEMOVIS), a preparation technique based on cutting thin sections with a knife, as an alternative to FIB-milling. CEMOVIS sections also sustain damage, including compression, shearing and cracks. However, samples can be sectioned in series, producing many orders of magnitude more imageable area compared to lamellae making CEMOVIS an alternative to FIB-milling with distinct advantages. Using 2-dimensional template matching on images of CEMOVIS sections of Saccharomyces cerevisiae cells, we reconstructed the 60S ribosomal subunit at near-atomic resolution, demonstrating that, in many regions of the sections, the molecular structure of these subunits is largely intact, comparable to FIB-milled lamellae.

biophysics↗

bioRxiv2024

Two-dimensional condensates of HRS drive the assembly of flat clathrin lattices on endosomes

In cells, the curved clathrin structures in vesicle budding are well characterized, while the flat ones remain poorly understood. We reconstituted the flat assembly of ESCRT-0 protein HRS and clathrin onto lipid membranes in vitro. HRS was found to form protein condensates. These condensates spread as a two-dimensional layer on negatively charged membranes and promoted the assembly of clathrin into a flat coat. Correlative cryo-tomography of HRS-labeled endosomes revealed a pure hexagonal lattice, consistent with flat clathrin structures. Cholesterol enhanced HRS recruitment to the membrane both in cells and in supported bilayers. Furthermore, cholesterol promoted the phase separation of HRS onto membranes, which in turn concentrated cholesterol underneath. This positive feedback promoted the formation of HRS-clathrin microdomains that sorted reconstituted ubiquitinated cargoes. Altogether, our results show that the unique architecture of ESCRT-0 is assembled by the two-dimensional phase-separation of HRS which drives the assembly of flat clathrin coats.

biochemistry↗

bioRxiv2024

Large transient assemblies of Apaf1 constitute the apoptosome in cells

Upon cell death signals, the apoptotic protease-activating factor Apaf1 and cytochrome c interact to form the apoptosome complex. The apoptosome is crucial for mitochondrial apoptosis, as it activates caspases that dismantle the cell. However, the assembly mechanism and appearance of the apoptosome in vivo remain unclear. We show that upon onset of apoptosis, Apaf1 molecules accumulate into multiple foci per cell. Disassembly of the foci is linked to survival of the cell. Structurally, Apaf1 foci resemble organelle-sized, cloud-like assemblies. Foci form upon specific molecular interactions with cytochrome c and depending on procaspase-9. We propose that Apaf1 foci correspond to the apoptosome in cells. Transientness and ultrastructure of Apaf1 foci suggest that the dynamic spatiotemporal organisation of apoptosome components regulates progression of apoptosis.

cell biology↗

bioRxiv2023

The punctate localisation of the yeast sterol transporter Ysp2p is determined by three dimerisation interfaces in its C-terminus

Sterol lipids traffic between intracellular compartments by vesicular and non-vesicular routes. Sterol traffic from the plasma membrane to the endoplasmic reticulum (ER), so-called retrograde traffic, particularly depends on a non-vesicular mechanism, being transported by the ubiquitous family of Lipid transfer proteins Anchored at Membrane contact sites (LAMs, also called GRAMD1/Asters in humans, VASt in plants). LAMs are similar to many lipid transfer proteins in that they localise to membrane contact sites and carry lipids between two organelles. In yeast, the major LAM active at ER-plasma membrane contact sites is Ysp2p, which has a uniquely punctate distribution in the cortical ER. Here, we have comprehensively dissected how Ysp2p achieves its distinctive punctate localisation. We show that the PHGRAM domain of Ysp2p has membrane binding properties similar to its human counterpart GRAMD1B, but that this is not important for punctate localisation of Ysp2p. Instead, all regions necessary for the punctate localisation of Ysp2p at membrane contacts are present in [~]200 residues at the C-terminus of Ysp2p, with a critical region being a small y-sheet that we predict homodimerises. We also study the role of punctate localisation of Ysp2 in its function in retrograde sterol traffic, and show that function does not require the punctate localisation, but instead requires a polybasic region adjacent to the sterol transfer domain. Finally, to investigate the interaction of the polybasic region with the plasma membrane, we examine contacts populated by the Ysp2 C-terminus by electron tomography, and find that they consist of generic cortical ER.

cell biology↗

bioRxiv2022

Supramolecular architecture of the ER-mitochondria encounter structure in its native environment

The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis which rely on lipid exchange via membrane contact sites, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the ER-mitochondria encounter structure ERMES, a four-subunit complex of unclear stoichiometry and architecture. We determined the molecular organization of ERMES within cells using integrative structural biology, combining quantitative live-imaging, cryo-correlative microscopy, subtomogram averaging and molecular modeling. ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three lipid-binding SMP domains arranged in zig-zag fashion. Our molecular model of ERMES reveals an unconventional restrained pathway for lipids. These findings resolve a supramolecular architecture controlling interorganelle lipid fluxes.

cell biology↗

bioRxiv2022

Ultrastructure of COPII vesicle formation characterised by correlative light and electron microscopy.

Traffic of proteins out of the endoplasmic reticulum (ER) is driven by the COPII coat, a layered protein scaffold that mediates the capture of cargo proteins and the remodelling of the ER membrane into spherical vesicular carriers. Although the components of this machinery have been genetically defined, and the mechanisms of coat assembly extensively explored in vitro, understanding the physical mechanisms of membrane remodelling in cells remains a challenge. Here we use correlative light and electron microscopy (CLEM) to visualize the nanoscale ultrastructure of membrane remodelling at ER exit sites (ERES) in yeast cells. Using various COPII mutants, we have determined the broad contribution that each layer of the coat makes in membrane remodelling. Our data suggest that inner coat components define the radius of curvature whereas outer coat components facilitate membrane fission. The organization of the coat in conjunction with membrane biophysical properties determine the ultrastructure of vesicles and thus the efficiency of protein transport.

cell biology↗

bioRxiv2021

The architecture of Cidec-mediated interfaces between lipid droplets

Lipid droplets (LDs) are intracellular organelles responsible for storing surplus energy as neutral lipids. Their size and number vary enormously (1). In white adipocytes, they reach up to 100 {micro}m in size, occupying >90% of the cell. Cidec, which is strictly required for the formation of such large LDs, is concentrated at interfaces between adjacent LDs and facilitates the directional flux of neutral lipids from the smaller to the larger LD (2-7). However, the mechanism of lipid transfer is unclear, in part because the architecture of interfaces between LDs has remained elusive. Here we visualised interfaces between LDs by electron cryo-tomography (cryo-ET) and analysed the kinetics of lipid transfer by quantitative live fluorescence microscopy (FM). We show that transfer occurs through closely apposed intact monolayers, is slowed down by increasing the distance between the monolayers and follows exponential kinetics suggesting a pressure-driven mechanism. We thus propose that unique architectural features of LD-LD interfaces are mechanistic determinants of neutral lipid transfer.

cell biology↗

bioRxiv2021

Ultrastructural plasma membrane asymmetries in tension and curvature promote yeast cell fusion

Cell-cell fusion is central to the process of fertilization for sexual reproduction. This necessitates the remodeling of peri-cellular matrix or cell wall material and the merging of plasma membranes. In walled fission yeast S. pombe, the fusion of P and M cells during sexual reproduction relies on the fusion focus, an actin structure that concentrates glucanase-containing secretory vesicles for local cell wall digestion necessary for membrane fusion. Here, we present a correlative light and electron microscopy (CLEM) quantitative study of a large dataset of 3D tomograms of the fusion site, which revealed the ultrastructure of the fusion focus as an actin-containing, vesicle-dense structure excluding other organelles. Unexpectedly, the data revealed asymmetries between the two gametes: M-cells exhibit a taut and convex plasma membrane that progressively protrudes into P-cells, which exhibit a more slack, wavy plasma membrane. These asymmetries are relaxed upon plasma membrane fusion, with observations of ramified pores that may result from multiple initiations or inhomogeneous expansion. We show that P-cells have a higher exo-to endocytosis ratio than M-cells, and that local reduction in exocytosis abrogates membrane waviness and compromises cell fusion significantly more in P-than M-cells. Reciprocally, reduction of turgor pressure specifically in M-cells prevents their protrusions into P-cells and delays cell fusion. Thus, asymmetric membrane conformations, which result from differential turgor pressure and exocytosis/endocytosis ratios between mating types, favor cell-cell fusion.

cell biology↗

bioRxiv2021

The molecular infrastructure of glutamatergic synapses in the mammalian forebrain

Glutamatergic synapses form the vast majority of connections within neuronal circuits, but how these subcellular structures are molecularly organized within the mammalian brain is poorly understood. Conventional electron microscopy using chemically fixed, metal- stained tissue has identified a proteinaceous, membrane-associated thickening called the postsynaptic density (PSD). Here, we combined mouse genetics and cryo-electron tomography to determine the 3D molecular architecture of fresh isolated and anatomically intact synapses in the adult forebrain. The native glutamatergic synapse did not consistently show a higher density of proteins at the postsynaptic membrane thought to be characteristic of the PSD. Instead, a synaptoplasm consisting of cytoskeletal elements, macromolecular complexes and membrane-bound organelles extended throughout the pre- and post- synaptic compartments. Snapshots of active processes gave insights into membrane remodeling processes. Clusters of up to 60 ionotropic glutamate receptors were positioned inside and outside the synaptic cleft. Together, these information-rich tomographic maps present a detailed molecular framework for the coordinated activity of synapses in the adult mammalian brain.

neuroscience↗

bioRxiv2020

The supramolecular landscape of growing human axons

During brain development, human axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly inaccessible to imaging at molecular resolution in a near-native context. Here we apply cryo-correlative light microscopy and electron tomography to growing axonal tracts from human cerebral organoids. Our data reveal a wealth of structural details on the arrangement of macromolecules, cytoskeletal components, and organelles in elongating axon shafts. In particular, the intricate shape of the endoplasmic reticulum is consistent with its role in fulfilling the high demand for lipid biosynthesis to support growth. Furthermore, the scarcity of ribosomes within the growing shaft suggests limited translational competence during expansion of this compartment. These data provide an unprecedented resource and reveal a molecular architecture that helps explain the unique biology of growing human axons.

cell biology↗

bioRxiv2020

Cargo crowding drives sorting stringency in COPII vesicles

Accurate maintenance of organelle identity in the secretory pathway relies on retention and retrieval of resident proteins. In the endoplasmic reticulum (ER), secretory proteins are packaged into COPII vesicles that largely exclude ER residents and misfolded proteins by mechanisms that remain unresolved. Here we combined biochemistry and genetics with correlative light and electron microscopy (CLEM) to explore how selectivity is achieved. Our data suggest that vesicle occupancy dictates ER retention: in the absence of abundant cargo, non-specific bulk flow increases. We demonstrate that ER leakage is influenced by vesicle size and cargo occupancy: overexpressing an inert cargo protein, or reducing vesicle size restores sorting stringency. We propose that cargo recruitment into vesicles creates lumenal steric pressure that drives selectivity. Sorting stringency is thus an emergent property of the biophysical process of cargo enrichment into a constrained spherical membrane-bound carrier. SummaryCombining correlative light and electron microscopy with yeast genetics and biochemistry, Gomez-Navarro, Melero and colleagues show that cargo recruitment into a constrained COPII vesicle restricts bulk flow, thereby contributing to sorting stringency and ER quality control.

cell biology↗

bioRxiv2019

Closed mitosis requires local disassembly of the nuclear envelope

At the end of mitosis, eukaryotic cells must segregate both copies of their replicated genome into two new nuclear compartments (1). They do this either by first dismantling and later reassembling the nuclear envelope in a so called \"open mitosis\", or by reshaping an intact nucleus and then dividing into two in a \"closed mitosis\" (2, 3). However, while mitosis has been studied in a wide variety of eukaryotes for over a century (4), it is not known how the double membrane of the nuclear envelope is split into two at the end of a closed mitosis without compromising the impermeability of the nuclear compartment (5). In studying this problem in the fission yeast Schizosaccharomyces pombe, a classical model for closed mitosis (5), we use genetics, live cell imaging and electron tomography to show that nuclear fission is achieved via local disassembly of the nuclear envelope (NE) within the narrow bridge that links segregating daughter nuclei. In doing so, we identify a novel inner NE-localised protein Les1 that restricts the process of local NE breakdown (local NEB) to the bridge midzone and prevents the leakage of material from daughter nuclei. The mechanics of local NEB in a closed mitosis closely mirror those of NEB in open mitosis (3), revealing an unexpectedly deep conservation of nuclear remodelling mechanisms across diverse eukaryotes.

cell biology↗

bioRxiv2019

Rod-shaped tricalbins contribute to PM asymmetry at curved ER-PM contact sites

Lipid flow between cellular organelles occurs via membrane contact sites that form dynamic conduits. Extended-synaptotagmins, known as tricalbins in yeast, mediate lipid transfer between the endoplasmic reticulum (ER) and plasma membrane (PM). How these proteins regulate the membrane architecture to transport lipids across the aqueous space between bilayers remains unknown. Using correlative microscopy, electron cryo-tomography and high-throughput genetics we address this interplay of architecture and function in budding yeast. We find that ER-PM contacts are diverse in protein composition and membrane morphology, not in intermembrane distance. In situ cryo-EM of tricalbins reveals their molecular organisation that suggests an unexpected structural framework for lipid transfer. Genetic analysis identifies functional redundancies, both for tricalbin domains and cellular lipid routes, and points to tricalbin function in maintenance of PM asymmetry. These results uncover a modularity of molecular and structural functions of tricalbins, and of their roles within the network of cellular lipid fluxes.

cell biology↗

bioRxiv2019

Multiplexed electron microscopy by fluorescent barcoding allows screening for ultrastructural phenotype

Genetic screens performed using high-throughput fluorescent microscopes have generated large datasets that have contributed many insights into cell biology. However, such approaches typically cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) is not subject to this resolution limit, generating detailed images of cellular ultrastructure, but requires time consuming preparation of individual samples, limiting its throughput. Here we overcome this obstacle and describe a robust method for screening by high-throughput electron microscopy. Our approach uses combinations of fluorophores as barcodes to mark the genotype of each cell in mixed populations, and correlative light and electron microscopy to read the fluorescent barcode of each cell before it is imaged by electron microscopy. Coupled with an easy-to-use software workflow for correlation, segmentation and computer image analysis, our method allows to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate the method on several organelles with samples that each contain up to 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher-throughput, and can be utilized in multiple ways to enable electron microscopy to become a powerful screening methodology.

cell biology↗

bioRxiv2018

Molecular and topological reorganizations in mitochondrial architecture interplay during Bax-mediated steps of apoptosis

During apoptosis, Bcl-2 proteins such as Bax and Bak mediate the release of pro-apoptotic proteins from the mitochondria by clustering on the outer mitochondrial membrane and thereby permeabilizing it. However, it remains unclear how outer membrane openings form. Here, we combined different correlative microscopy and electron cryo-tomography approaches to visualize the effects of Bax activity on mitochondria in human cells. Our data show that Bax clusters localize near outer membrane ruptures of highly variable size. Bax clusters contain structural elements suggesting a higher-order organization of their components. Furthermore, unfolding of inner membrane cristae is coupled to changes in the supramolecular assembly of ATP synthases, particularly pronounced at membrane segments exposed to the cytosol by ruptures. Based on our results, we propose a comprehensive model in which molecular reorganizations of the inner membrane and sequestration of outer membrane components into Bax clusters interplay in the formation of outer membrane ruptures.

cell biology↗

bioRxiv2017

The contributions of the actin machinery to endocytic membrane bending and vesicle formation

Branched and crosslinked actin networks mediate cellular processes that move and shape membranes. To understand how actin contributes during the different stages of endocytic membrane reshaping, we analysed deletion mutants of yeast actin network components using a hybrid imaging approach that combines live imaging with correlative microscopy. We could thereby temporally dissect the effects of different actin network perturbations, revealing distinct stages of actin-based membrane reshaping. Our data show that initiation of membrane bending requires the actin network to be physically linked to the plasma membrane and to be optimally crosslinked. Once initiated, the membrane invagination process is driven by nucleation and polymerization of new actin filaments, independently of the degree of cross-linking and unaffected by a surplus of actin network components. A key transition occurs 2 seconds before scission when the filament nucleation rate drops. From that time point on, invagination growth and vesicle scission are driven by an expansion of the assembled actin network. The expansion is sensitive to the amount of filamentous actin and its crosslinking. Our results suggest that the mechanism by which actin reshapes the membrane adapts to force requirements that vary during the progress of endocytosis.

cell biology↗