Traffic

Spatial and temporal tracking of fluorescent proteins in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active fluorescent proteins (FPs) fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae. SIGNIFICANCE STATEMENT- Quantitative endocytic assays are required to characterize factors that regulate both ligand-dependent and constitutive endocytosis. - We optimize fluorogen-activating proteins (FAPs) technology for use as a live cell imaging probe in yeast that fluoresces in the far-red range for quantitative endocytosis assays. - The FAP tools and approaches generated will facilitate quantitative endocytic and protein recycling assays for yeast cell biologists.

SYNOPSISThe AP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. Here, a synthetic reporter is presented that allows convenient monitoring of AP-3 traffic, and direct screening or selection for mutants with defects in the pathway. The reporter can be assayed by fluorescence microscopy or in liquid or agar plate formats and is adaptable to high-throughput screening. SUMMARYAP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. In mammals, mutations in AP-3 cause Hermansky-Pudlak Syndrome type 2, cyclic neutropenias, and a form of epileptic encephalopathy. In budding yeast, AP-3 carries cargo directly from the trans-Golgi to the lysosomal vacuole. Despite the pathways importance and its discovery two decades ago, rapid screens and selections for AP-3 mutants have not been available. We now report GNSI, a synthetic, genetically encoded reporter that allows rapid plate-based assessment of AP-3 functional deficiency, using either chromogenic or growth phenotype readouts. This system identifies defects in both the formation and consumption of AP-3 carrier vesicles and is adaptable to high-throughput screening or selection in both plate array and liquid batch culture formats. Episomal and integrating plasmids encoding GNSI have been submitted to the Addgene repository.

In long-lived cells such as neurons, proteostasis involves the regulated degradation and replacement of proteins to ensure their quality and appropriate abundance. Synaptic vesicle (SV) protein turnover in neurons is important for controlling the SV pool size to maintain appropriate levels of neurotransmission; yet, it is incompletely understood, partly due to limited tools for quantifying protein turnover in vivo. We present ARGO (Analysis of Red-Green Offset), a fully genetically encoded, ratiometric fluorescence imaging method that visualizes and quantifies protein turnover with subcellular resolution in vivo. ARGO is inexpensive, modular, and scalable for use in genetically tractable experimental organisms. Using ARGO, we examine the turnover of Synaptogyrin/SNG-1, an evolutionarily conserved, integral SV protein, in C. elegans neurons. We show that the SNG-1 turnover rate is consistent across presynapses within a single neuron but varies between neuron classes. Notably, we find SNG-1 and can exist in two distinct, non-intermixing populations within each presynapse. Further, we present an initial mutant analysis of uba-1, the sole E1 ubiquitin ligase in C. elegans, showing that we can detect slowed SNG-1 turnover even though steady-state SNG-1 abundance is not increased compared to wild-type. These results provide new hints for the regulation of SV pool size. Significance Statement- In long-lived cells, a proteins rates of synthesis and degradation together determine its abundance, yet regulation of protein turnover is largely unknown due to the lack of simple methods for in vivo quantification. - Using C. elegans, the authors develop ARGO, a genetically encoded, microscopy approach that quantifies a proteins turnover. Results suggest that synaptic vesicle protein Synaptogyrin/SNG-1 can partition into two presynaptic pools with distinct half-lives. - This provides a powerful tool to study protein turnover, reveals an unexpected complexity in SNG-1 turnover, and lays the groundwork for future investigations of synaptic vesicle protein compartmentalization, sorting, and degradation.

Mon1a has been shown to function in the endolysosomal pathway functioning in the Mon1-Ccz1 complex and it also acts in the secretory pathway where it interacts with dynein and affects ER to Golgi traffic. Here we show that Mon1a is also required for maintenance of the Golgi apparatus. We identified the F-BAR protein FCHO2 as a Mon1a-interacting protein by both yeast two-hybrid analysis and co-immunoprecipitation. siRNA-dependent reductions in Mon1a or FCHO2 resulted in Golgi fragmentation. Membrane trafficking through the secretory apparatus in FCHO2-depleted cells was unaltered, however, reduction of FCHO2 affected the uniform distribution of Golgi enzymes necessary for carbohydrate modification. Fluorescence recovery after photobleaching analysis showed that the Golgi ministacks in Mon1a- or FCHO2-silenced cells did not exchange resident membrane proteins. The effect of FCHO2 silencing on Golgi structure was partially cell cycle-dependent and required mitosis-dependent Golgi fragmentation, whereas the effect of Mon1a-silencing on Golgi disruption was not cell cycle-dependent. mCherry-FCHO2 transiently colocalized on Golgi structures independent of Mon1a. These findings suggest that Mon1a has functions throughout the secretory pathway including interacting with dynein at the ER-Golgi interface in vesicle formation and then interacting with FCHO2 at the Golgi to generate lateral links between ministacks, thus creating Golgi ribbons.

This study aims to demonstrate how PTP-3 regulates SYD-2 to control UNC-104-mediated axonal transport. UNC-104 is the C. elegans homolog of kinesin-3 KIF-1A known for its fast shuttling of STVs (synaptic vesicle protein transport vesicles) in axons. SYD-2 is the homolog of liprin- in C. elegans known to directly regulate UNC-104 as well as being a substrate of LAR PTPR (leukocyte common antigen-related (LAR) protein tyrosine phosphatase (PTP) transmembrane receptor) with PTP-3 as the closest homolog in C. elegans. CoIP assays revealed increased interaction between UNC-104 and SYD-2 in lysates from ptp-3 knockout worms. Intramolecular FRET analysis revealed that SYD-2 predominantly exists in an open conformation state in ptp-3 mutants. These assays also revealed that non-phosphorylatable SYD-2 (Y741F) exists predominately in folded conformations while phosphomimicking SYD-2 (Y741E) exists predominantly in open conformations. In ptp-3 mutants, SNB-1 cargo accumulates in soma while at the same time UNC-104 motors increasingly cluster along initial segments of axons. Interestingly, the unc-104 gene is downregulated in ptp-3 mutants that might explain the vesicle retention phenotype. More strikingly, the few visibly moving motors and STVs were overly active in neurons of these mutants. We propose a model in which the lack of PTP-3 promotes increased open conformations of SYD-2 that in turn facilitates UNC-104/SYD-2 interactions boosting motor and STVs moving speeds.

All endo- and exocytosis in the African trypanosome Trypanosoma brucei occurs at a single subdomain of the plasma membrane. This subdomain, the flagellar pocket, is a small vase-shaped invagination containing the root of the cells single flagellum. Several cytoskeleton-associated multiprotein complexes are coiled around the neck of the flagellar pocket on its cytoplasmic face. One of these, the hook complex, was proposed to affect macromolecule entry into the flagellar pocket lumen. In previous work, knockdown of the hook complex component TbMORN1 resulted in larger cargo being unable to enter the flagellar pocket. In this study, the hook complex component TbSmee1 was characterised in bloodstream form Trypanosoma brucei and was found to be essential for cell viability. TbSmee1 knockdown resulted in flagellar pocket enlargement and impaired access to the flagellar pocket membrane by surface-bound cargo, similar to depletion of TbMORN1. Unexpectedly, inhibition of endocytosis by knockdown of clathrin phenocopied TbSmee1 knockdown, suggesting that endocytic activity itself is a prerequisite for the entry of surface-bound cargo into the flagellar pocket. SummaryCharacterisation of the essential trypanosome protein TbSmee1 suggests that endocytosis is required for flagellar pocket access of surface-bound cargo.

The vesicle-tethering exocyst complex is a key regulator of cell polarity. The subunit Exo70 is required for the targeting of the exocyst complex to the plasma membrane. While the N-terminus of Exo70 is important for its regulation by GTPases, the C-terminus binds to PI(4,5)P2 and Arp2/3. Here, we compare N- and C-terminal tagged Exo70 with respect to subcellular localization, dynamics and function in cell membrane expansion. Using high-resolution imaging, we determined the spatial distribution and dynamics in different sub-compartments of un-polarized and polarized cells. With lattice light-sheet microscopy, we show that HaloTag-Exo70, but not Exo70-HaloTag, promotes the outgrowth of filopodia-like structures from the axon of hippocampal neurons. Fluorescence lifetime imaging of sfGFP-Exo70 and molecular modeling results suggest that the assembly of sfGFP-Exo70 with the exocyst complex is reduced. This is supported by single particle tracking data showing higher mobility of N- than C-terminal tagged Exo70 at the plasma membrane. The distinct spatiotemporal properties of N-terminal tagged Exo70 were correlated with pronounced filopodia formation in unpolarized cells and neurons, a process that is less reliant on exocyst complex formation. We therefore propose that N-terminal tagging of Exo70 shifts its activity to processes that are less exocyst-dependent. Why it mattersIn the life sciences, the high-resolution visualization of processes in living cells is of great interest. To tag proteins, they are fused with fluorescent proteins, usually at the N- or C-terminus of the amino acid sequence. We show here that the position of the tag alters the function and interaction of Exo70, a polypeptide involved in vesicle fusion, but also in membrane bending and expansion. Using state-of-the-art microscopic techniques, single particle localization and tracking, fluorescence lifetime imaging microscopy and co-localization in combination with modeling, we conclude that N-terminal tagging of Exo70 impairs its assembly with the exocyst complex and instead promotes the interaction of free Exo70 with the actin skeleton, which favors controlled membrane expansion into filipodia.

Membrane contact sites (MCSs) are areas of close proximity between organelles that allow the exchange of material, among other roles. The endoplasmic reticulum (ER) has MCSs with a variety of organelles in the cell. MCSs are dynamic, responding to changes in cell state, and are therefore best visualized through inducible labeling methods. However, existing methods typically distort ER-MCSs, by expanding contacts or creating artificial ones. Here we describe a new method for inducible labeling of ER-MCSs using the Lamin B receptor (LBR) and a generic anchor protein on the partner organelle. Termed LaBeRling, this versatile, one-to-many approach allows labeling of different types of ER-MCSs (mitochondria, plasma membrane, lysosomes, early endosomes, lipid droplets and Golgi), on-demand, in interphase or mitotic cells. LaBeRling is non-disruptive and does not change ER-MCSs in terms of the contact number, extent or distance measured; as determined by light microscopy or a deep-learning volume electron microscopy approach. We applied this method to study the changes in ER-MCSs during mitosis and to label novel ER-Golgi contact sites at different mitotic stages in live cells.

The internal organisation of the cell depends on tethers at destination organelles to selectively capture incoming transport vesicles to facilitate SNARE-mediated fusion. The golgin long coiled-coil proteins function as tethers that contributes to this specificity at the Golgi (1). Golgin-97, golgin-245 and GCC88 golgins of the trans-Golgi capture vesicles derived from endosomes, which serve to recycle the critical Golgi machinery required to deliver lysosomal hydrolases and to maintain exocytosis. Retrograde trafficking from endosomes to the trans-Golgi network (TGN) is a complex process that involves the sorting of transmembrane cargo proteins into distinct transport vesicles by adaptors from multiple pathways. The content of these distinct vesicles, which golgin they target and the factors that mediate this targeting are not well understood. The major challenges that have limited advances in these areas is the transient nature of vesicle tethering, and the redundancies in their mechanisms that confound experimental dissection. To gain better insight into these problems, we performed organelle proteomics using the Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation (LOPIT-DC) method on a system in which an ectopic golgin causes vesicles to accumulate in a tethered state (2). By incorporating Bayesian statistical modelling into our analysis (3), we determined that over 45 transmembrane proteins and 51 peripheral membrane proteins of the endosomal network are on vesicles captured by golgin-97, including known cargo and components of the clathrin/AP-1, retromer-dependent and -independent transport pathways. We also determined a distinct class of vesicles shared by golgin-97, golgin-245 and GCC88 that is enriched in TMEM87A, a multi-pass transmembrane protein of unknown function that has previously been implicated in endosome-to-Golgi retrograde transport (4). Finally, we categorically demonstrate that the vesicles that these golgins capture are retrograde transport vesicles based on the lack of enrichment of lysosomal hydrolases in our LOPIT-DC data, and from correlative light electron tomography images of spherical vesicles captured by golgin-97. Together, our study demonstrates the power of combining LOPIT-DC with Bayesian statistical analysis in interrogating the dynamic spatial movement of proteins in transport vesicles.

In yeast and humans, the conserved DENN-domain (Differentially Expressed in Normal and Neoplastic tissue) protein Avl9 is thought to play roles in membrane traffic and secretion, but its precise function remains poorly defined. Since DENN-containing proteins are associated with Rab GTPase function, we sought to understand Avl9 function in the context of Rab regulation. Here, we show that Avl9 localizes to peripheral punctae that are consistent with secretory vesicles. Moreover, we demonstrate genetic interactions and co-localization between Avl9 and numerous Rabs in the secretory and endosomal pathways, suggesting a potential function at the interface of secretion and recycling. Consistent with this role, avl9{Delta} results in defective recycling of the endosomal cargo Snc1 but does not alter plasma membrane delivery of an endocytosis-defective Snc1EN- mutant, suggesting that Avl9 is not directly involved in secretory traffic from the TGN to the plasma membrane. The avl9{Delta} recycling defect is exacerbated by the additional loss of RCY1 or SNX4, but not VPS35. Each of these three genes contributes to a distinct endosomal recycling pathway, indicating that Avl9 acts in conjunction with multiple recycling pathways. Summary StatementIn this study, Rioux et al. describe a role for the DENN domain protein Avl9, previously thought to regulate secretion, as a novel factor involved in recycling of cargos from endosomal compartments.

Directional dendritic transport of late endosomes retrogradely towards the soma is required for fusion with lysosomes and for degradation in the soma. Both dendritic motility of late endosomes and somatic degradation require RAB7A. Similarly, interference with dynein function reduces motility of late endosomes and results in degradative failure. Blocking dynein function also impairs normal dendrite growth, suggesting that motility of late endosomes and/or lysosomes might be required for dendrite growth. RAB7A and dynein are mechanistically linked via RILP which is a dynein-interacting RAB7A effector. RILP also binds the late endosome-lysosome fusion tether HOPS. In non-neuronal cells, downregulation of RILP leads to impaired degradation due to deficiencies in late endosome transport and fusion defects with lysosomes. In this work, we express a separation-of-function mutant of RAB7A (RAB7A-L8A) incapable of RILP binding. Based on the results in non-neuronal cells, we hypothesized that both endosome motility and degradation in neurons depended on RILP. Our data in cultured rat and mouse hippocampal neurons of both sexes suggest that endogenous RILP is a functional RAB7A-dependent dynein adaptor for late endosome motility in dendrites. Interestingly, it also promotes endosome carrier formation. As a consequence of late endosome transport inhibition, degradative cargos are not cleared normally from dendrites in RAB7A-L8A. Surprisingly, lysosomal fusion and somatic degradation do not require RAB7A-RILP interactions. Despite the normal degradation, dendrite arborization is impaired in RAB7A-L8A expressing neurons, demonstrating that dendrite morphology defects are separable from degradation blockade. This indicates that normal dendrite growth/maintenance is dependent on sustained RAB7A/RILP-dependent LE transport. Significance StatementDendrite growth requires membrane trafficking, but the roles of individual compartments and regulators are not well established. Stunted dendrite growth is often associated with endolysosomal traffic jams and degradation block. In contrast, our work reveals a requirement for transport of late endosomes to support dendrite growth independently of late endosomes as carriers of degradative cargos to the lysosome for degradation.

Kinesin-5 motors play essential roles in spindle apparatus assembly during cell division, by generating forces to establish and maintain the spindle bipolarity essential for proper chromosome segregation. Kinesin-5 is largely conserved structurally and functionally in model eukaryotes, but its role is unknown in the Plasmodium parasite, an evolutionarily divergent organism with several atypical features of both mitotic and meiotic cell division. We have investigated the function and subcellular location of kinesin-5 during cell division throughout the Plasmodium berghei life cycle. Deletion of kinesin-5 had little visible effect at any proliferative stage except sporozoite production in oocysts, resulting in a significant decrease in the number of motile sporozoites in mosquito salivary glands, which were able to infect a new vertebrate host. Live-cell imaging showed kinesin-5-GFP located on the spindle and at spindle poles during both atypical mitosis and meiosis. Fixed-cell immunofluorescence assays revealed kinesin-5 co-localized with -tubulin and centrin-2 and a partial overlap with kinetochore marker NDC80 during early blood stage schizogony. Dual-colour live-cell imaging showed that kinesin-5 is closely associated with NDC80 during male gametogony, but not with kinesin-8B, a marker of the basal body and axonemes of the forming flagella. Treatment of gametocytes with microtubule-specific inhibitors confirmed kinesin-5 association with nuclear spindles and not cytoplasmic axonemal microtubules. Altogether, our results demonstrate that kinesin-5 is associated with the spindle apparatus, expressed in proliferating parasite stages, and important for efficient production of infectious sporozoites.

The contractile vacuole complex (CVC) is a dynamic and morphologically complex membrane organelle, comprised of a large vesicle (bladder) linked with a tubular reticulum (spongiome). CVCs provide key osmoregulatory roles across diverse eukaryotic lineages, but probing the mechanisms underlying the structure and function is hampered by the limited tools available for in vivo analysis. In the experimentally tractable ciliate Tetrahymena thermophila, we describe four proteins that, as endogenously tagged constructs, localize specifically to distinct CVC zones. The DOPEY homolog Dop1p and the CORVET subunit Vps8Dp localize both to the bladder and spongiome but with different local distributions that are sensitive to osmotic perturbation, while the lipid scramblase Scr7p co-localizes with Vps8Dp. The H+- ATPase subunit Vma4 is spongiome-specific. The live imaging permitted by these probes revealed dynamics at multiple scales including rapid exchange of CVC-localized and soluble protein pools vs. lateral diffusion in the spongiome, spongiome extension and branching, and CVC formation during mitosis. While the association with DOP1 and VPS8D implicate the CVC in endosomal trafficking, both the bladder and spongiome are isolated from bulk endocytic input. Summary statementIn the ciliate Tetrahymena thermophila, four proteins are shown to provide markers for different zones of the contractile vacuole complex. They shed light on its formation and maintenance by enabling in vivo analysis of its dynamics.

This study reveals new insights into the role of the Adaptor protein (AP-3) complex in dense core vesicle function. Despite numerous studies, an existing knowledge lacuna in the role of AP-3 in DCV function prompted us to delve deeper. Advanced microscopy and biochemical analysis revealed compromised DCV exocytosis in AP-3-depleted PC12 cells and C. elegans. AP-3 depletion altered the size and positioning of DCVs. Golgi defects and RUSH (Retention under Selective Hook) substantiated the role of AP-3 in trans-Golgi DCV budding. Proteomics revealed the loss of specific known and putative novel DCV proteins, which were mislocalized and rerouted to lysosomes in AP-3-depleted cells. Bioinformatics, Proximity ligation assays and Co-immunoprecipitation identified interactions of mislocalized proteins with AP-3 subunit. These findings corroborated with functional defects in granule maturation, release modes, Zinc and neurotransmitter mobilisation. Our study highlights the complexity of the AP-3 complex in regulating DCV function and its importance in vesicle transport in neurons and neuroendocrine cells. SummaryThis work reveals the critical role of AP-3 complex in DCV function, highlighting its impact on DCV exocytosis, positioning, and trans-Golgi budding. This study identifies Dlk1 as a novel DCV cargo.

RAB GTPases are the most abundant family of small GTPases and regulate multiple aspects of membrane trafficking events, from cargo sorting to vesicle budding, transport, docking, and fusion. To regulate these processes, RABs are tightly regulated by guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs). Activated RABs recruit effector proteins that regulate trafficking. Identifying RAB-associated proteins has proven to be difficult because their association with interacting proteins is often transient. Recent advances in proximity labeling approaches that allow for the covalent labeling of neighbors of proteins of interest now permit the cataloging of proteins in the vicinity of RAB GTPases. Here, we report APEX2 proximity labeling of 23 human RABs and their neighboring proteomes. We have used bioinformatic analyses to map specific proximal proteins for an extensive array of RAB GTPases, and RAB localization can be inferred from their adjacent proteins. Focusing on specific examples, we identified a physical interaction between RAB25 and DENND6A, which affects cell migration. We also show functional relationships between RAB14 and the EARP complex, or between RAB14 and SHIP164 and its close ortholog UHRF1BP1. Our dataset provides an extensive resource to the community and helps define novel functional connections between RAB GTPases and their neighboring proteins.

Macrophages clear invading pathogens by phagocytosis. Phagocytosis is a complex mechanism involving the local expansion of the membrane, cytoskeletal remodeling, and the delivery of phagosomal proteins to the nascent phagosomes. However, the organelle trafficking events underlying this are largely unclear. Here, we show in human blood monocyte-derived macrophages that TRPML1, a calcium channel involved in the phagocytic process, is delivered to phagosomes in Syntaxin 3-positive vesicles. Syntaxin 3 is a SNARE protein previously shown to mediate the secretion of IL-6 by macrophages. Total Internal Reflection Microscopy (TIRF) revealed that Syntaxin 3 positive compartments carry TRPML1 to pseudopodia for focal exocytosis at the nascent phagosomes during E. coli uptake. Using siRNA knockdown, we show that both Syntaxin 3 and TRPML1 are required for E. coli uptake. Moreover, using TRPML1 agonists we show that increased TRPML1 activity leads to increased E. coli uptake, whereas calcium chelation decreased intracellular E. coli load. Understanding the membrane trafficking pathways is critical for understanding how macrophages clear invading pathogens. Key findingsO_LISyntaxin 3 positive vesicles are delivered at the plasma membrane site of phagocytosis. C_LIO_LISyntaxin 3-positive vesicles carry TRPML1 to pseudopodia. C_LIO_LIBacterial phagocytosis correlates with Syntaxin 3 and TRPML1 expression levels. C_LIO_LIBacterial phagocytosis depends on calcium flux through TRPML1. C_LIO_LISyntaxin-3 vesicles carry the cytokine interleukin-6. C_LI

The primary cilium is an antenna-like organelle that plays a crucial role in development and homeostasis. Its growth and functions depend on the transport of cargo from the cilia base to the tip by intraflagellar transport (IFT) proteins and kinesin-2 motors. Primary cilia exhibit great variation in morphology and function across cell types and organisms. This diversity is thought to be conferred by the modulation of IFT by additional factors. However, examples of such non-canonical regulators, such as kinesin motors distinct from kinesin-2, are sparse. Thus, the involvement of non-canonical ciliary kinesins in intraciliary transport is unclear. Here, we show that a poorly characterized member of the kinesin-3 family, KIF14, plays a prominent role in primary cilia trafficking in human cells. Using live cell imaging and expansion microscopy, we demonstrate that KIF14 depletion leads to impaired IFT. Furthermore, using TIRF microscopy we show that the motility of KIF14 and its effects on cilia trafficking rely on the motor domain of KIF14, which drives the processive runs along the ciliary axoneme in cells and in vitro, in co-operation with the C-terminal CC1 domain. Finally, we demonstrate that C-terminal truncations of KIF14, including patient mutation Q1380x, disrupt IFT by causing traffic jam-like accumulations of ciliary components in the distal part of the primary cilia, leading to bulged tips of cilia. In summary, our data exemplify the role of a non-canonical ciliary kinesin in the regulation of ciliary trafficking and suggest a new paradigm for how kinesin-related trafficking defects may contribute to the pathology of disorder with a ciliary contribution.

Although the structure of the exocyst has been successfully resolved by cryo-electron microscopy, multiple studies showed that exocyst function requires the transient interaction with additional proteins. Unfortunately, the exocyst-interacting network could not be collectively reconstituted, challenging the understanding of how the exocyst complex is coordinated within the network of proteins involved in exocytosis. In a previous work, we described an approach that combines Protein interactions from Imaging Complexes after Translocation (PICT) and centroid localization analysis of diffraction-limited fluorescence signals to estimate the distance between a labelled protein and a spatial reference. This approach allows resolving the spatial organisation of protein interactions directly in living cells, both for intra-complex (i.e. between exocyst subunits) and inter-complex (i.e. between exocyst and transient binding proteins) interactions. In this chapter, we present the protocol to reproduce the sample preparation and image acquisition for PICT experiments. We also describe the computational image analysis pipeline to estimate the distance in PICT experiments. As illustration of the approach, we measure the distance from the spatial reference where the exocyst is anchored to 1) an intra-complex interaction (i.e. Sec5 exocyst subunit) and 2) an inter-complex interaction (Sec2, a guanyl-nucleotide exchange factor mediating vesicle tethering).

Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample [~]4.5x. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three-dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have catalogued 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the outer centriolar plaque and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the outer centriolar plaque until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an association with the outer centriolar plaque during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date, and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology. IMPACT STATEMENTUsing ultrastructure-expansion microscopy we explore the fundamental cell biology of malaria parasites, providing new insights into processes including establishment of cell polarity and organelle fission.

The canonical function of the Golgi-associated retrograde protein (GARP) complex is the tethering of transport carriers. GARP belongs to the complexes associated with tethering containing helical rods (CATCHR) family and is a hetero-tetrameric complex consisting of the subunits Vps51, Vps52, Vps53 and Vps54. How the activity of GARP is regulated and if it possesses other functions besides tethering remains largely unknown. Here we identify the GARP subunit Vps53 as a novel regulatory target of the S. cerevisiae AMP kinase (AMPK) homolog Snf1. We find that Vps53 is both an in vivo and in vitro target of Snf1 and show that phosphorylation depends on the nature and quantity of the available carbon source. Phosphorylation of Vps53 does not affect the canonical trafficking pathway, but results in altered mitochondrial dynamics and the formation of a previously unknown contact site between the Golgi apparatus and mitochondria, termed GoMiCS. Our results provide an example of a subunit of a CATCHR complex with a constitutive function in membrane trafficking and an inducible role in organelle contact site formation. We anticipate our results to be the starting point for the characterization of this novel contact site.