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bioRxiv2025

IntAct-U-ExM: Ultrastructure Expansion microscopy of actin networks via an internally-tagged actin

Expansion microscopy (ExM) has revolutionized super-resolution imaging in cell biology due to its simple and inexpensive workflow. The use of ExM has revealed several novel insights into the nanoscale architectures of cellular protein complexes, especially the microtubule cytoskeleton in model and non-model systems. Despite tremendous progress in expansion microscopy protocols that preserve cellular ultrastructure (U-ExM), compatible probes for imaging actin isoforms with U-ExM are still lacking and have hindered the study of diverse actin isoforms and networks across model systems. Here, we use IntAct, an internally tagged actin that incorporates into cellular actin networks, to develop and optimize U-ExM of diverse actin network types in both yeast and mammalian cells. Using expression of ALFA-tagged IntAct variants in yeast and mammalian cells, we show robust visualization of actin patches, cables, and rings in yeast and diverse actin networks such as actin cortex, stress fibers, filopodia, lamellipodium in mammalian cells at improved resolution. We also detect transient nuclear actin filaments using IntAct-U-ExM underscoring the advantages offered by our approach to image understudied actin structures. Overall, we demonstrate the effectiveness of IntAct-U-ExM for performing super-resolution imaging of various actin structures in an isoform-specific manner and highlight the potential of IntAct to study the nanoscale organization of diverse actin cytoskeletal networks across species. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/654030v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@169cd82org.highwire.dtl.DTLVardef@1334af3org.highwire.dtl.DTLVardef@7d9d43org.highwire.dtl.DTLVardef@dfc50b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Complete absence of GLUT1 does not impair human terminal erythroid differentiation

The Glucose transporter 1 (GLUT1) is one of the most abundant proteins within the erythrocyte membrane and is required for glucose and dehydroascorbic acid (Vitamin C precursor) transport. It is widely recognized as a key protein for red cell structure, function, and metabolism. Previous reports highlighted the importance of GLUT1 activity within these uniquely glycolysis-dependent cells, in particular for increasing antioxidant capacity needed to avoid irreversible damage from oxidative stress in humans. However, studies of glucose transporter roles in erythroid cells are complicated by species-specific differences between humans and mice. Here, using CRISPR-mediated gene editing of immortalized erythroblasts and adult CD34+ hematopoietic progenitor cells, we generate committed human erythroid cells completely deficient in expression of GLUT1. We show that absence of GLUT1 does not impede human erythroblast proliferation, differentiation, or enucleation. This work demonstrates for the first-time generation of enucleated human reticulocytes lacking GLUT1. The GLUT1-deficient reticulocytes possess no tangible alterations to membrane composition or deformability in reticulocytes. Metabolomic analyses of GLUT1-deficient reticulocytes reveal hallmarks of reduced glucose import, downregulated metabolic processes and upregulated AMPK-signalling, alongside alterations in antioxidant metabolism, resulting in increased osmotic fragility and metabolic shifts indicative of higher oxidant stress. Despite detectable metabolic changes in GLUT1 deficient reticulocytes, the absence of developmental phenotype, detectable proteomic compensation or impaired deformability comprehensively alters our understanding of the role of GLUT1 in red blood cell structure, function and metabolism. It also provides cell biological evidence supporting clinical consensus that reduced GLUT1 expression does not cause anaemia in GLUT1 deficiency syndrome. Key PointsO_LIGLUT1 knockout does not affect erythroid differentiation and minimally impacts reticulocyte membrane composition C_LIO_LIMetabolic adaptation facilitates reticulocyte tolerance of GLUT1 absence C_LI

cell biology↗

bioRxiv2020

Protein kinase Cϵ activation induces EHD-dependent degradation and downregulation of KATP channels: Implications for glucose stimulated insulin secretion

Pancreatic {beta}-cells have the unique ability to couple glucose metabolism to insulin secretion. This capacity is generally attributed to the ability of ATP to inhibit KATP channels, and the consequent {beta}-cell membrane depolarization and excitation. This notion has recently been challenged by a study which demonstrated that high glucose (HG) downregulates the cell surface KATP channels, and thereby leads to {beta}-cell depolarisation and excitation. The authors attributed the downregulation to HG-induced protein kinase C (PKC) activation and the consequent increase in channel endocytosis. This interpretation, however, is inconsistent with our previous findings that PKC activation does not affect endocytosis. To address this controversy, we revisited the problem: we have used cell biological and electrophysiological approaches combined with the pharmacological activator of PKC, PMA (phorbol 12-myristate 13-acetate). We first confirm that PKC does not play a role in KATP channel endocytosis; instead, it downregulates the channel by promoting lysosomal degradation coupled with reduced recycling. We then show that (i) mutation of the dileucine motif (355LL356) in the C-terminal domain of the Kir6.2 subunit of the KATP channel complex prevents lysosomal degradation; (ii) lysosomal targeting is mediated by the EHD (Eps15 homology domain- containing) proteins; and (iii) the PKC isoform responsible for channel degradation is PKC{varepsilon}. Taken together with the published data, we suggest that HG promotes {beta}-cell excitability via two mechanisms: ATP-dependent channel inhibition and ATP-independent, PKC{varepsilon}-dependent channel degradation. The results likely have implications for glucose induced biphasic insulin secretion.

cell biology↗

bioRxiv2023

The Dilute domain of Canoe is not essential for Canoe's role in linking adherens junctions to the cytoskeleton but contributes to robustness of morphogenesis

Robust linkage between cell-cell adherens junctions and the actomyosin cytoskeleton allows cells to change shape and move during morphogenesis without tearing tissues apart. The multidomain protein Drosophila Canoe and its mammalian homolog Afadin are critical for this linkage, and in their absence many events of morphogenesis fail. To define underlying mechanisms, we are taking Canoe apart, using Drosophila as our model. Canoe and Afadin share five folded protein domains, followed by a large intrinsically disordered region. The largest of these folded domains is the Dilute domain, which is found in Canoe/Afadin, their paralogs, and members of the MyosinV family. To define the roles of Canoes Dilute domain we have combined biochemical, genetic and cell biological assays. Use of the AlphaFold tools revealed the predicted structure of the Canoe/Afadin Dilute domain, providing similarities and contrasts with that of MyosinV. Our biochemical data suggest one potential shared function: the ability to dimerize. We next generated Drosophila mutants with the Dilute domain cleanly deleted. Surprisingly, these mutants are viable and fertile, and Canoe{Delta}DIL protein localizes to adherens junctions and is enriched at junctions under tension. However, when we reduce the dose of Canoe{Delta}DIL protein in a sensitized assay, it becomes clear it does not provide full wildtype function. Further, canoe{Delta}DIL mutants have defects in pupal eye development, another process that requires orchestrated cell rearrangements. Together, these data reveal the robustness in AJ-cytoskeletal connections during multiple embryonic and postembryonic events, and the power of natural selection to maintain protein structure even in robust systems.

cell biology↗

bioRxiv2022

Calcium flux through ER-TGN contact sites facilitates cargo export

Ca2+ influx into the trans-Golgi Network (TGN) promotes secretory cargo sorting by the Ca2+-ATPase SPCA1 and the luminal Ca2+ binding protein Cab45. Cab45 oligomerizes upon a local Ca2+ influx, and Cab45 oligomers sequester and separate soluble secretory cargo from the bulk flow of proteins in the TGN. However, how this Ca2+ flux into the lumen of the TGN is achieved remains elusive, as the cytosol has a very low steady-state Ca2+ concentration. The TGN forms membrane contact sites (MCS) with the Endoplasmic Reticulum (ER), whereby the close apposition of the two organelles allows the protein-mediated exchange of molecular species such as lipids. Here we show that the TGN export of Cab45 clients requires the integrity of ER-TGN MCS and IP3R-dependent Ca2+ fluxes in the MCS, suggesting Ca2+ transfer between these organelles. Using an MCS-targeted Ca2+ FRET sensor module, we measure the Ca2+ flow in these sites in real-time. These data show for the first time that ER-TGN MCS facilitates Ca2+ transfer required for SPCA1-dependent cargo sorting and export from the TGN, thus solving a fundamental question in cell biology. SummaryThe current study demonstrates that the trafficking of COMP and LyzC relies on Ca2+ flux between the endoplasmic reticulum (ER) and trans-Golgi Network (TGN). This process requires the activity of IP3 receptors, present in ER membranes, and depends on the integrity of the membrane contact site between these two organelles.

cell biology↗

bioRxiv2021

Benchmarking a highly selective USP30 inhibitor for enhancement of mitophagy and pexophagy.

The deubiquitylase USP30 is an actionable target considered for treatment of conditions associated with defects in the PINK1/Parkin pathway leading to mitophagy. These include Parkinsons disease and pulmonary fibrosis. We provide a detailed cell biological characterisation of a benzenesulphonamide molecule, compound 39, that has previously been reported to inhibit USP30 in an in vitro enzymatic assay. The current compound offers increased selectivity over previously described inhibitors. It enhances mitophagy and generates a signature response for USP30 inhibition following mitochondrial depolarisation. This includes enhancement of TOM20 and SYNJ2BP ubiquitylation and phosphoubiquitin accumulation, alongside increased mitophagy. In dopaminergic neurons, generated from Parkinsons disease patients carrying loss of function Parkin mutations, compound 39 could significantly restore mitophagy to a level approaching control values. USP30 is located on both mitochondria and peroxisomes and has also been linked to the PINK1 independent pexophagy pathway. Using a fluorescence reporter of pexophagy expressed in U20S cells, we observe increased pexophagy upon application of compound 39 that recapitulates the previously described effect for USP30 depletion. This provides the first pharmacological intervention with a synthetic molecule to enhance peroxisome turnover.

cell biology↗

bioRxiv2024

Spatiotemporal proteomic profiling of cellular responses to NLRP3 agonists

Nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 3 (NLRP3) is an innate immune sensor that forms an inflammasome in response to various cellular stressors. Gain-of-function mutations in NLRP3 cause autoinflammatory diseases and NLRP3 signalling itself exacerbates the pathogenesis of many other human diseases. Despite considerable therapeutic interest, the primary drivers of NLRP3 activation remain controversial due to the diverse array of signals that are integrated through NLRP3. Here, we mapped subcellular proteome changes to lysosomes, mitochondrion, EEA1-positive endosomes, and Golgi caused by the NLRP3 inflammasome agonists nigericin and CL097. We identified several common disruptions to retrograde trafficking pathways, including COPI and Shiga toxin-related transport, in line with recent studies. We further characterized mouse NLRP3 trafficking throughout its activation using temporal proximity proteomics, which supports a recent model of NLRP3 recruitment to endosomes during inflammasome activation. Collectively, these findings provide additional granularity to our understanding of the molecular events driving NLRP3 activation and serve as a valuable resource for cell biological research. We have made our proteomics data accessible through an open-access Shiny browser to facilitate future research within the community, available at: https://harperlab.connect.hms.harvard.edu/inflame/. We will display anonymous peer review for this manuscript on pubpub.org (https://harperlab.pubpub.org/pub/nlrp3/) rather than a traditional journal. Moreover, we invite community feedback on the pubpub version of this manuscript, and we will address criticisms accordingly.

cell biology↗

bioRxiv2026

A screen for adherens junction proteins regulating collective cell migration and testis morphogenesis reveals important roles for the Rab GAP RN-tre and the kinase Par-1

Animal tissues have diverse architectures and cell behaviors across the epithelial-mesenchymal spectrum. Cell adhesion mediated by classical cadherins is foundational. Cadherins nucleate complexes of dozens of proteins connecting junctions to the cytoskeleton and signaling downstream. Many junctional proteins are well-studied in epithelia, but less is known about roles during mesenchymal migration. The nascent myotubes of the pupal Drosophila testis provide an excellent model for N-cadherin mediated mesenchymal migration. We combined a proximity proteomics dataset of adherens junction proteins in mammalian epithelial cells with genome-wide shRNA libraries knocking down Drosophila genes to begin to define the subset of junctional proteins important in mesenchymal migration. While N-cadherin is predominant, E-cadherin plays a supporting role. Surprisingly, several proteins with key roles in epithelial morphogenesis, including Afadins homolog Canoe, ZO-1s homolog Polychaetoid, and Par3s homolog Bazooka play at most modest roles. Twenty-two genes with diverse cell biological roles had strong to moderate defects in testis morphogenesis. These will provide a community resource. We followed up two. The kinase Par-1 is important for migration and gap closure, with knockdown phenotypes paralleling those of myosin. The Rab GAP RN-tre does not have roles until after migration and works in parallel with N-cadherin during testis spiralization.

cell biology↗

bioRxiv2024

The evolutionary origins of the lysosome-related organelle sorting machinery reveal fundamental homology in post-endosome trafficking pathways

The major organelles and pathways of the endomembrane system were in place by the time of the last eukaryotic common ancestor (LECA) ([~]1.5 billion years ago) and their acquisition were defining milestones during the process of eukaryogenesis itself. Comparative cell biology and evolutionary analyses show multiple instances of homology in the protein machinery controlling distinct inter-organelle trafficking routes. Resolving these homologous relationships allows us to explore processes underlying the emergence of new cellular compartments, infer ancestral states pre-dating LECA, and can even provide insight into the process of eukaryogenesis itself. Here we undertake a molecular evolutionary analysis, including providing a transcriptome of the jakobid flagellate Reclinomonas americana, exploring the origins of the machinery responsible for the biogenesis of lysosome-related organelles, the so-called Biogenesis of Lysosome-related Organelle Complexes (BLOCs 1,2, and 3). This pathway has been studied only in animals and is not considered a feature of the basic eukaryotic cell plan. We show that this machinery, and by inference the corresponding sorting pathway, was likely in place prior to the divergence of eukaryotes and is found in a much more diverse array of eukaryotes than is currently assumed. As such, this sorting pathway is likely an underappreciated facet of broader eukaryotic cellular function. Moreover, we resolve multiple points of ancient homology between all three BLOCs and other post-endosomal retrograde trafficking machinery (BORC, CCZ1/MON1, and a newly identified relationship with HOPS/CORVET) offering a mechanistic and evolutionary unification of these trafficking pathways. Overall, this study provides a comprehensive account of the rise of the LRO biogenesis machinery from prokaryotic origins to current eukaryotic diversity, Asgard archaea to animals, integrating it into the larger mechanistic framework describing endomembrane evolution.

cell biology↗

bioRxiv2022

TGF-β and BMP subfamily pathways in zebrafish spermatogonial niche: A83-01 and DMH1 inhibitor effects in spermatogonial self-renewal and differentiation

This study unravels the roles of TGF-{beta} (Transforming growth factor-{beta}) superfamily signaling pathway in the zebrafish spermatogonial activity (self-renewal vs. differentiation) by combining ex vivo and specific pathway inhibitors approaches. The TGF-{beta} superfamily signaling pathway is subdivided into TGF-{beta} and Bone morphogenetic proteins (BMP) subfamilies, and is ubiquitous among metazoans, regulating several biological processes, including spermatogenesis. In this study, we evaluated the function of the TGF-{beta} and BMP subfamily pathways in the zebrafish spermatogonial niche using A83-01 and DMH1 inhibitors, respectively. Our results showed that A83-01 potentiated the follicle-stimulating hormone (Fsh) effects on zebrafish spermatogenesis, reducing type A undifferentiated spermatogonia and increasing differentiated spermatogonia (type Adiff and type B spermatogonia) after 7 days of culture. In agreement with histomorphometrical data, the mRNA levels of dazl (marker of spermatogonial differentiation) and pro-differentiation growth factors, such as igf3 and insl3, were significantly augmented following A83-01. For the BMP signaling pathway, exposure to DMH1 inhibitor showed opposite effects as compared to TGF-{beta} superfamily signaling pathway inhibitor. Histomorphometrical analysis demonstrated an accumulation of type A undifferentiated spermatogonia, while the frequency of differentiated spermatogonia was significantly reduced following co-treatment of DMH1 with Fsh after 7 days of culture. To support this data, expression analysis revealed that BMP signaling pathway inhibitor also decreased the testicular mRNA levels of dazl, igf3 and insl3 when compared to control incubation (Fsh). In conclusion, our study demonstrated that TGF-{beta} and BMP subfamily pathways exert a role in zebrafish spermatogonial niche with antagonistic functions for the spermatogonia fate. The TGF-{beta} subfamily pathway is involved with spermatogonial self-renewal and inhibition of differentiation, whereas the BMP subfamily pathway promotes spermatogonial differentiation. These findings are not only relevant to understanding stem cell biology, but may also be useful in several in vitro assays, promoting control of self-renewal and differentiation by potentially directing these processes.

cell biology↗

bioRxiv2023

Membrane binding of endocytic myosin-1s is inhibited by a class of ankyrin repeat proteins

Myosin-1s are monomeric actin-based motors that function at membranes. Myo1 is the single myosin-1 isoform in Schizosaccharomyces pombe that works redundantly with Wsp1-Vrp1 to activate the Arp2/3 complex for endocytosis. Here, we identified Ank1 as an uncharacterized cytoplasmic Myo1 binding partner. We found that in ank1{Delta} cells, Myo1 dramatically redistributed from endocytic patches to decorate the entire plasma membrane and endocytosis was defective. Biochemical analysis and structural predictions suggested that the Ank1 ankyrin repeats bind the Myo1 lever arm and the Ank1 acidic tail binds the Myo1 TH1 domain to prevent TH1-dependent Myo1 membrane binding. Indeed, Ank1 over-expression precluded Myo1 membrane localization and recombinant Ank1 blocked purified Myo1 liposome binding in vitro. Based on biochemical and cell biology analyses, we propose budding yeast Ank1 and human OSTF1 are functional Ank1 orthologs and that cytoplasmic sequestration by small ankyrin repeat proteins is a conserved mechanism regulating myosin-1s in endocytosis. SummaryFission yeast long-tailed myosin-1 binds Ank1. Ank1 ankyrin repeats associate with the Myo1 lever arm and Ank1 acidic tail binds the Myo1 TH1 domain to inhibit Myo1 membrane binding. Ank1 orthologs exists in budding yeast (Ank1) and humans (OSTF1).

cell biology↗

bioRxiv2020

The Formin Inhibitor, SMIFH2, Inhibits Members of the Myosin Superfamily

The small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It was selected in a chemical screen as a compound inhibiting formin-driven actin polymerization in vitro, but not polymerization of pure actin, and found to be active against several types of formins from different species (Rizvi et al., 2009). Here, in experiments with cultured fibroblasts, we found that SMIFH2 inhibits retrograde flow of myosin 2 filaments and contraction of stress fibers. We further checked the effect of SMIFH2 on non-muscle myosin 2A and skeletal muscle myosin 2 in vitro and found that SMIFH2 inhibits myosin ATPase activity and ability to translocate actin filaments in the in vitro motility assay. While inhibition of myosin 2A in vitro required somewhat higher concentration of SMIFH2 than inhibition of retrograde flow and stress fiber contraction in cells, inhibition of several other non-muscle myosin types, e.g. mammalian myosin 10, Drosophila myosin 7a and Drosophila myosin 5 by SMIFH2, was equally or more efficient than inhibition of formins. Since actin polymerization and myosin contractility are linked in many cytoskeleton processes, additional careful analysis is needed in each case when function of formins was proposed solely on the basis of experiment with SMIFH2.

cell biology↗

bioRxiv2021

De novo disease-associated mutations in KIF1A dominant negatively inhibit axonal transport of synaptic vesicle precursors

KIF1A is a kinesin superfamily molecular motor that transports synaptic vesicle precursors in axons. Mutations in Kif1a lead to a group of neuronal diseases called KIF1A-associated neuronal disorder (KAND). KIF1A forms a homodimer and KAND mutations are mostly de novo and autosomal dominant; however, it is not known whether the function of wild-type KIF1A is inhibited by disease-associated KIF1A when they are dimerized. No reliable in vivo model systems to analyze the molecular and cellular biology of KAND caused by loss of function mutations have been developed; therefore, here, we established Caenorhabditis elegans models for KAND using CRISPR/cas9 technology and analyzed defects in axonal transport. In the C. elegans models, heterozygotes and homozygotes exhibited reduced axonal transport phenotypes. Suppressor screening using the disease model worm identified a mutation that recovers the motor activity of disease-associated human KIF1A. In addition, we developed in vitro assays to analyze the motility of single heterodimers composed of wild-type KIF1A and disease-associated KIF1A. Disease-associated KIF1A significantly inhibited the motility of wild-type KIF1A when heterodimers were formed. These data indicate the molecular mechanism underlying the dominant nature of de novo KAND mutations. Significance StatementKIF1A is a molecular motor that transports synaptic vesicle precursors in axons. Recent studies have identified many KIF1A mutations in congenital neuropathy patients; however, the molecular mechanism of pathogenesis remains largely elusive. This study established loss of function models for KIF1A-associated neuronal disorder (KAND) in Caenorhabditis elegans to analyze the molecular and cell biology of the disease in vivo. Genetic screening using the disease model could find a mutation that recovers the motor activity of disease-associated KIF1A. This study also established in vitro single-molecule assays to quantitatively analyze the effect of KAND mutations when mutant KIF1A forms heterodimers with wild-type KIF1A. Our findings provide a foundation for future genetic screening and for drug screening to search for KAND treatments.

cell biology↗

bioRxiv2026

Membrane lipid composition and endocytosis modulate Wingless release from secreting cells

Wnts are secreted signalling molecules that regulate development and adult homeostasis. Most Wnts carry a lipid moiety that must be shielded from the aqueous environment. In the secretory pathway, this is achieved by a hydrophobic tunnel in Wntless, a multipass transmembrane protein. However, the Wnt lipid moiety must be released from Wntless before Wnts can engage with Frizzled receptors on receiving cells. Here we address the cell biological basis of Wnt-Wntless dissociation, using as a model the secretion of Drosophila Wingless in wing primordia. Super-resolution microscopy shows that Wingless first reaches the apical surface before being re-internalized to reach, without Wntless, specialized Rab7/Rab4-positive endosomes. From there Wingless traffics to the basolateral membrane where it can engage with glypicans to form a basolateral gradient. Acute inhibition of endocytosis, either with a temperature-sensitive dynamin mutant or a novel optogenetic means of inhibiting clathrin, leads to apical Wingless release in abnormal punctae devoid of Wntless, suggesting that Wingless-Wntless dissociation commences at the apical surface, perhaps because of a distinct lipid composition there. Indeed, similar looking punctae are produced upon genetic abrogation of the ceramide synthase Schlank, specifically in Wingless-producing cells. These punctae resemble insoluble aggregates that form in vitro upon detergent removal. Accordingly, punctae formation can be prevented by shielding the Wingless lipid, in vivo with excess Dally-like protein (Dlp) or in vitro with liposomes. Our results show that membrane lipid composition modulates the orderly transfer of Wingless lipid from Wntless to the inner endosomal surface thus preventing aggregation and ensuring seamless secretion in the basolateral space.

cell biology↗

bioRxiv2024

Mechanism and regulation of cargo entry into the Commander recycling pathway

Commander is a multiprotein complex that orchestrates endosomal recycling of diverse integral cargo proteins and in humans is required for normal skeletal, brain, kidney, and cardiovascular development. While the structure of this complex has recently been described, the central question of how cargo proteins are selected for entry into the Commander recycling pathway remains unclear. Here using recombinant protein reconstitution and in silico predictions we identify the evolutionary conserved mechanism through which the unstructured carboxy-terminal tail of the integral protein adaptor sorting nexin-17 (SNX17) directly binds to the Retriever sub-complex of Commander. SNX17 adopts an autoinhibited conformation where its carboxy-terminal tail occupies the cargo binding groove. Competitive cargo binding overcomes this autoinhibition, promoting SNX17 endosomal residency and the release of the carboxy tail for Retriever association. Using molecular cell biology and high-resolution microscopy, we establish the central importance of SNX17-Retriever association in the handover of integrin and lipoprotein receptor cargoes into pre-existing endosomal retrieval sub-domains for entry into the recycling pathway. In describing the principal mechanism of cargo entry into the Commander recycling pathway we provide key insight into the function and regulation of this evolutionary conserved sorting complex.

cell biology↗

bioRxiv2026

Global mapping of protein localization in vegetative Dictyostelium discoideum by subcellular spatial proteomics

Dictyostelium discoideum is a genetically tractable amoebozoan model organism widely used to study conserved eukaryotic cellular processes, yet a comprehensive map of its subcellular proteome has been lacking. Here, we combined subcellular fractionation with label-free mass spectrometry to resolve a global protein localization map for D. discoideum. We detected 6,337 proteins (~50% of the predicted proteome) and generated protein abundance profiles across 10 subcellular fractions. Using a curated marker set and a support vector machine classifier with a median cutoff, we assigned high-confidence localizations to 3,169 proteins across 17 subcellular compartments. Independent validation using sequence-based targeting predictions strongly supported the compartment assignments. Together, this study provides the first global subcellular localization map of the Dictyostelium proteome, establishing a foundational resource for functional, cell biological, and evolutionary analyses. We are currently preparing manuscripts that further analyze (i) the membrane trafficking system of Dictyostelium, including detailed investigation of the contractile vacuole, Jotnarlogs, and patchy proteins, and (ii) the composition of the Dictyostelium peroxisome, with a focus on the subcellular localization of sterol biosynthesis enzymes. Investigators interested in using these data are encouraged to contact us prior to publication to avoid overlap and to facilitate coordinated and collaborative use of this resource.

cell biology↗

bioRxiv2021

Expansion microscopy facilitates quantitative super-resolution studies of cytoskeletal structures in kinetoplastid parasites

Expansion microscopy (ExM) has become a powerful super-resolution method in cell biology. It is a simple, yet robust approach, which does not require any instrumentation or reagents beyond those present in a standard microscopy facility. In this study, we used kinetoplastid parasites Trypanosoma brucei and Leishmania major, which possess a complex, yet well-defined microtubule-based cytoskeleton, to demonstrate that this method recapitulates faithfully morphology of structures as previously revealed by a combination of sophisticated electron microscopy (EM) approaches. Importantly, we also show that due to rapidness of image acquisition and 3D reconstruction of cellular volumes ExM is capable of complementing EM approaches by providing more quantitative data. This is demonstrated on examples of less well-appreciated microtubule structures, such as the neck microtubule of T. brucei or the pocket, cytosolic, and multivesicular tubule-associated microtubules of L. major. We further demonstrate that ExM enables identifying cell types rare in a population, such as cells in mitosis and cytokinesis. 3D reconstruction of an entire volume of these cells provided details on morphology of the mitotic spindle and the cleavage furrow. Finally, we show that established antibody markers of major cytoskeletal structures function well in ExM, which together with the ability to visualize proteins tagged with small epitope tags will facilitate studies of the kinetoplastid cytoskeleton.

cell biology↗

bioRxiv2023

Characterizing Human KIF1Bβ Motor Activity by Single-Molecule Motility Assays and Caenorhabtidis elegans Genetics.

The axonal transport of synaptic vesicle precursors relies on KIF1A and UNC-104 ortholog motors. In mammals, KIF1B{beta} is also responsible for the axonal transport of synaptic vesicle precursors. Mutations in KIF1A and KIF1B{beta} lead to a wide range of neuropathies. While previous studies have revealed the biochemical, biophysical and cell biological properties of KIF1A, and its defects in neurological disorders, the fundamental properties of KIF1B{beta} remain elusive. In this study, we determined the motile parameters of KIF1B{beta} through single-molecule motility assays. Additionally, we established simple methods for testing the axonal transport activity of human KIF1B{beta} using Caenorhabditis elegans genetics. Taking advantage of these methods, we demonstrated that these assays enable the detection of reduced KIF1B{beta} activities both in vitro and in vivo, that is caused by a disease-associated mutation.

cell biology↗