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Toxoplasma gondii is a single-celled eukaryotic parasite with prolific invasion capability. The parasite uses an apical complex comprised of proteinaceous structures and secretory organelles to efficiently enter host cells. As a result, the apical complex remains a vital structure of interest, with many studies dedicated to understanding its protein organization. One such protein is the motor Myosin H (MyoH), which is indispensable for parasite motility and host cell invasion. Given the small size of the complex, roughly a diffraction-limited volume in the visible, high-resolution techniques are required to make precise determinations of protein organization. In this work, we use 3D single-molecule localization microscopy in both traditionally fixed and gel-expanded parasites to localize the indispensable motor Myosin H within the apical complex. Labeling of the N- and C-terminus of MyoH in fixed parasites resolved the orientation of the motor protein in the apical complex, showing the motor head radially exterior to the tail. Two-color imaging of MyoH with tubulin in fixed parasites allowed for localization of the MyoH termini relative to the conoid, a barrel of tubulin-based fibers in the apical complex and showed the MyoH tail toward the interior face of the conoid and the head at the conoid exterior. Gel expansion showed improved labeling density for both tubulin and MyoH but altered MyoH localization, highlighting the nuanced effects of gel expansion on protein organization. Statement of SignificanceThis work employs 3D single-molecule super-resolution microscopy to provide quantitative physical analysis of the spatial organization of a vital myosin motor, MyoH, in the model apicomplexan parasite Toxoplasma gondii. While previous studies have provided high-resolution views of the parasites invasion machinery, MyoH has remained elusive at the nanoscale. We resolved differences in radial organization between the N- and C-termini of the motor, thus determining the orientation of the protein in the apical space. Two-color imaging revealed the organization of the motor in the greater context of the parasites invasion complex. 3D single-molecule imaging in gel-expanded samples revealed an increase in labeling efficiency but perturbed localization of only the MyoH C-terminus, highlighting the nuanced effects of gel expansion on protein organization.

Heterotrimeric kinesin 2 is the canonical motor protein for anterograde intraflagellar transport (IFT), driving movement of protein complexes towards the tip of cilia and flagella. Here, we show that all members of the Euglenozoa group lack genes for heterotrimeric kinesins and instead possess a variable number of genes for two homodimeric kinesins termed KIN2A and KIN2B. When expressed in vitro, both Trypanosoma brucei kinesins form homodimers and move processively along brain microtubules, KIN2A being faster than KIN2B. Studies in T. brucei and Leishmania mexicana show anterograde and retrograde IFT of both kinesins, with KIN2A travelling throughout the whole length of the flagellum, while KIN2B is concentrated at its base. In the proximal portion of the flagellum, most KIN2B molecules travel without IFT proteins, except for a few particles that are associated with IFT proteins and reach the tip. Surprisingly, the absence of KIN2A has mild effects on IFT and flagellum assembly, whereas KIN2B is essential for both. Investigation of trypanosome flagella deprived of KIN2B revealed that IFT proteins do not access these flagella but that KIN2A can still circulate. These results support a division-of-labour model where KIN2B is responsible for the import of IFT proteins while KIN2A is responsible for most of the anterograde transport.

Following ER Ca2+ depletion, Ca2+ release-activated Ca2+ (CRAC) channels are activated by STIM1 at ER-plasma membrane junctions. The restricted localization and low conductance of the CRAC channel (<40 fS) precludes single-channel recordings, limiting studies of CRAC channel gating. Here we describe an optical approach to characterize the gating of HaloTag-fused Orai1 channels labeled with JF646-BAPTA, a Ca2+-sensitive fluorescent dye. While Ca2+ influx through single channels generates fluorescence fluctuations, identifying true gating events is complicated by stochastic transitions of JF646-BAPTA to a non-fluorescent state. To overcome this, we combine TIRF microscopy with whole-cell voltage clamp to control the driving force for Ca2+ entry. We show the open channel intensity at -100 mV reflects Ca2+ saturation of the dyes on each channel, while the closed-channel intensity is defined by the fluorescence at +30 mV, where influx is absent. True gating events can be identified from transitions between the open- and closed-channel levels, distinguishing them from transitions to a non-fluorescent state. We describe the gating behavior of CRAC channels activated by STIM1 after store depletion. Dwell time distributions indicate at least two open and closed states with durations of 0.1 to several seconds, with most channels having an open probability of [&ge;]0.7. We also detect silent channels that colocalize with STIM1 but show no activity over tens of seconds, a population that would be undetectable by whole-cell electrophysiology alone. This method offers an approach to explore CRAC channel gating mechanisms and may be applicable to other Ca2+- permeable channels not amenable to patch-clamp techniques.

During maturation of a Golgi cisterna, multiple vesicular transport pathways recycle resident Golgi proteins. Recycling vesicles are captured by Golgi-associated tethers. To assign individual tethers to specific recycling pathways in Saccharomyces cerevisiae, we examined tether arrival and departure using kinetic mapping, and we examined tether function using an ectopic tether localization assay. Those approaches yielded mutually consistent results. Our analysis focused on two coiled coil golgin tethers and the multi-subunit tether GARP. At an intermediate stage of cisternal maturation, the golgin Sgm1 tethers proteins that follow an intra-Golgi recycling pathway dependent on COPI. At a late stage of cisternal maturation, GARP and the golgin Imh1 tether trans- Golgi network (TGN) proteins that follow an intra-Golgi recycling pathway dependent on the AP-1 and Ent5 clathrin adaptors. This involvement of GARP in intra-Golgi recycling had not previously been documented. Imh1 also tethers proteins that recycle from prevacuolar endosome compartments to the TGN. Our findings contribute to an integrated model of Golgi membrane traffic.

Kinesin-3 motor proteins are increasingly recognized for their important roles in cilia. The mammalian kinesin-3 motor KIF13B moves bidirectionally in primary cilia and regulates ciliary content, but its relationship to the intraflagellar transport (IFT) machinery is unclear. Here, we combine quantitative live-cell imaging with a new kymograph analysis based on dynamic mode decomposition (DMD) to separate mobile from immobile protein populations in primary cilia. This approach simplifies extraction of molecular velocities from kymographs and reveals that a KIF13B deletion mutant retaining only the motor domain and part of the forkhead-associated domain does not alter steady-state IFT velocity or frequency. However, when retrograde dynein-2 function is inhibited by Ciliobrevin D, both anterograde and retrograde IFT velocities decrease in parental cells, as expected, but remain unchanged in KIF13B mutant cells. Structured illumination, confocal, and STED microscopy further show that KIF13B localizes to the ciliary membrane and concentrates at the periciliary membrane region and the centriolar subdistal appendages, below the distal appendage marker FBF1. Our improved kymograph approach provides new insight into KIF13B ciliary function and simplifies the quantitative analysis of ciliary protein transport.

The endoplasmic reticulum (ER)-Golgi interface is a dynamic trafficking hub maintained in part by TANGO1, a scaffolding protein that coordinates proteins and membranes at ER exit sites (ERES). TANGO1 has two isoforms: TANGO1L, which has a lumenal SH3 domain, and TANGO1S, which lacks this domain but retains the transmembrane and cytoplasmic coiled-coil (CC), TEER, and PRD domains common to both forms. We showed previously that loss of both isoforms disrupts ER-Golgi organization more severely than TANGO1L loss alone, indicating TANGO1S is functional and can compensate. Here we dissect the role of each TANGO1 cytoplasmic domain in maintaining secretory pathway organisation by expressing TANGO1S domain-deletion mutants in TANGO1L-/S-knockout cells. We show that TANGO1 loss causes cis-Golgi vesiculation that cannot be rescued by TANGO1S, suggesting the lumenal domain of TANGO1L is essential in supporting Golgi architecture. Meanwhile, the TEER domain is essential for the organisation of the ER, whilst the TEER, CC2 and PRD domain are required for a defined ERGIC. All constructs partially rescue COPII recruitment. This study represents an advance towards a domain-level resolution of TANGO1S function. Summary statementIn this study we perform rescue experiments in TANGO1 knockout cells to dissect the role of the TANGO1 cytoplasmic domains in maintaining the ER-ERGIC-Golgi continuum.

Lipophagy is an important microautophagic process that degrades lipid droplets (LDs) to mobilize stored lipids as an energy source during nutrient starvation. However, the molecular mechanisms regulating lipophagy in response to nutrient starvation remain poorly understood. We found that budding yeast mutants defective in glycosylphosphatidylinositol (GPI) lipid remodeling exhibited aberrant accumulation of lipid droplets (LDs) and neutral lipids under glucose starvation. Our data suggest that the accumulation results from a failure of vacuolar liquid-ordered (Lo) domain-mediated lipophagy. Furthermore, we demonstrated that glycosylphosphatidylinositol-anchored proteins (GPI-APs) localize to vacuoles in response to glucose depletion and that a mutant defective in endocytosis has defects in both vacuolar Lo domain formation and lipophagy. These results imply that GPI lipid remodeling is required for Lo domain-mediated lipophagy upon glucose starvation. We propose that endocytosis functions to supply the lipid portion of GPI-APs, remodeled to C26 diacylglycerol, to the vacuolar membrane for Lo domain formation. Summary StatementOur data suggest that the endocytic transport of GPI-APs remodeled with C26 diacylglycerol to the vacuole is required for vacuolar Lo domain formation and subsequent lipophagy in response to glucose deprivation. This reveals the essential role of GPI lipid remodeling in ensuring lipophagy to adapt to changes in nutrient availability.

Synaptic vesicle proteins (SVPs) are synthesised in the neuronal soma trafficked as precursor synaptic vesicles (pre-SVs) on route to synapses. While pre-SVs are known to have heterogeneous protein composition and can co-traffic with lysosomal proteins. In this study, we assess the trafficking routes and kinetics of Synatobrevin-1 (SNB-1) released from the ER using the RUSH system in vivo in C. elegans touch receptor neurons. We showed that ER-released SNB-1 follows at least two temporally distinct trafficking routes. A predominantly anterogradely moving population of SNB-1 carrying vesicles appeared early, within 20 minutes of ER release in the axon without overlap with lysosomal proteins. Another SNB-1 population at 45 minutes post-ER release overlapped with endolysosomal compartments in both the cell body and the axon. Early SNB-1 carrying vesicles co-migrate with a transmembrane synaptic vesicle protein Synaptogyrin (SNG-1) and RAB-27 but fewer with RAB-3, suggesting that SVPs can be co-sorted into the same carriers prior to overlap with lysosomal proteins. The SV-lysosomal protein overlap occurs even when SNB-1 endocytosis on the plasma membrane is reduced in unc-11/ap180 mutants. Finally, we identified SAM-4/Myrlysin, a subunit of the BORC complex, as a regulator of both the trafficking kinetics of Synaptobrevin-1 intermediates and the cargo composition of pre-SVs. Loss of SAM-4 accelerated SV-lysosomal protein overlap and reduced co-transport of SNG-1 with SNB-1 in early pre-SVs in the axon. Together, these findings reveal heterogeneity in pre-SV biogenesis routes and identify SAM-4 as a key regulator of both the kinetics and cargo composition of synaptic vesicle precursors.

Extracellular vesicles are key intercellular messengers that modulate the function of target cells by carrying effectors, either at their surface or in their lumen. In the latter case, their action depends on the ability to deliver their content into the cytosol of target cells. How efficiently EVs deliver their content upon interaction with their target cell is thus a central question for understanding the functional impact of this mode of action. To address this question, signal-driven bimolecular interactions between two partners located respectively in the EV lumen and the target cell cytosol have become a widely used strategy to detect the cytosolic delivery EV content. However, the detection of cytosolic delivery with these assays was often tributary to the artificial enhancement of the fusion between EV and cell membranes, through for instance VSV-G fusogenic protein expression. Here we provide a robust and quantitative LUCiferase-based complementation assay (HiBiT/LgBiT), to quantify the Internalization and cytosolic Delivery of EV content: LUCID-EV. By optimizing the signal-to-noise ratio of the assay, the method for loading HiBiT fragment into EVs (fusion to a lipid-binding domain rather than to tetraspanins), and the intracellular position of LgBiT (associated to membranes), we could quantify cytosolic delivery from various non-VSV-G-expressing EVs into target immune dendritic cells. Importantly, this delivery did not involve the acidic late endosomes environment required for VSV-G-dependent EV cytosolic delivery. The limited efficacy of the process highlights the need for highly sensitive assays like the one described here. Further development of the LUCID-EV assay could help identifying EV/target cells pairs with enhanced cytosolic delivery properties and characterize the cellular route for delivery.

Subcellular organelles must undergo periodic fission to be evenly distributed during cell division. These division events are mediated by protein members of the dynamin family, including dynamin-related proteins. Protozoan parasites, including trypanosomatids such as Trypanosoma brucei, have several single-copy organelles, suggesting tightly regulated systems for organelle fission and segregation. However, trypanosomatid genomes typically encode only one dynamin-like protein (DLP), which in T. brucei has multiple roles including endocytosis and mitochondrial fission. How DLPs are recruited to different membranes, and how their fission activity is regulated, are unknown. We used tandem-affinity purification in the related trypanosomatid Crithidia fasciculata to identify interacting partners of DLP. Surprisingly, we found that CfDLP co-purified with multiple proteins predicted to localize to glycosomes, peroxisome-related glycolytic organelles. Using expansion microscopy, we confirmed the localization of CfDLP to glycosomes, specifically those that appear to be undergoing division. To see if changes in the levels of DLP could alter glycosome morphology, we conducted RNAi-mediated knockdown and inducible overexpression experiments in T. brucei. TbDLP knockdown causes subtle changes in glycosome size, while overexpression of TbDLP1 causes an increase cytoplasmic vesicles and altered permeability of glycosomal membranes. These results suggest that the multifunctional DLP of trypanosomatids plays a role in glycosome maintenance. Author SummaryTrypanosomatids are eukaryotic parasites that cause devastating diseases in humans and animals. Like all eukaryotic cells, they must maintain their subcellular compartments through organelle division and other membrane remodeling events. Dynamin-like proteins are enzymes that work with other proteins to apply mechanical force to membranes. The dynamin-like proteins of Trypanosoma brucei, the causative agent of human African trypanosomiasis, have been implicated in endocytosis and mitochondrial division, although how these activities are regulated is not known. We have used a model trypanosomatid, the mosquito parasite Crithidia fasciculata, to look for dynamin-interacting proteins. In addition to proteins of unknown function, we show that dynamin-like protein associates with proteins found on glycosomes, trypanosomatid-specific organelles that contain enzymes required for breakdown of sugars. Knockdown and overexpression of dynamin-like proteins in T. brucei causes changes in glycosomes, supporting a role in organelle maintenance. Dynamin-like proteins likely regulate organelle structure and function, allowing parasites to adapt to different energetic requirements during their life cycle.

At the early endosome, cargos are sorted into subdomains; receptors destined for recycling to the plasma membrane are sorted into tubulovesicular structures that undergo fission and release cargo-laden vesicles that traffic along microtubules. Although branched actin has been implicated in the establishment/maintenance of endosomal membrane subdomains, its role in cargo segregation, fission, and recycling has not been extensively studied. Using inhibitors of formin-and ARP2/3-mediated actin assembly, we show that branched actin, but not linear actin, is required for endosome fission and receptor recycling. To examine the spatial relationship between actin and cargo, we transfected cells with constitutively active RAB5 Q79L to generate enlarged endosomes and demonstrated that internalized transferrin localized to discrete endosomal regions adjacent to branched actin. ARP2/3 inhibition disrupted this organization, resulting in broader cargo distribution on the endosomal membrane and coalescence of degradative and retrieval subdomains. Consistent with impaired endosomal sorting and fission, branched actin inhibition led to cargo accumulation. Our findings identify ARP2/3-mediated branched actin as a key regulator of cargo segregation, subdomain maintenance, and fission at the early endosome.

Subcellular architecture is tightly controlled and contributes to the maintenance of cells homeostasis. Organelles are regulated in size, shape, number and position which respond to changes in extracellular environment. Lysosomes are of particular interest as they integrate various functions in the cells (nutrient sensing, metabolism, cell migration and adhesion), serving as signaling hubs. Their function is tightly linked to their subcellular position and deregulation of lysosome homeostasis leads to several diseases including cancer. Therefore, methods allowing precise analysis of organelle subcellular distribution can aid in fundamental, diagnostic and therapeutic approaches. Here, we provide a versatile image analysis pipeline using ImageJ and CellProfiler. This workflow allows to quantify subcellular lysosome distribution in living and fixed melanoma cells, and is applicable to other subcellular compartments and to various cell types.

Astrocytes directly influence neuronal survival and increasingly are understood to contribute to the progression of neurodegenerative diseases including Parkinsons disease (PD). Mitochondrial damage is a hallmark of PD pathology in both neurons and astrocytes. Damaged mitochondria are cleared by PINK1/Parkin-mediated mitophagy; loss-of-function mutations in either PINK1 or Parkin are sufficient to cause PD. Neuronal mitophagy is well-studied, but far less is known about how mitochondrial dysfunction in astrocytes affects neural health. While microglial release of pro-inflammatory cytokines has been shown to induce astrocytes to mount their own inflammatory response, we hypothesize that a more direct pathway is involved, and that mitochondrial damage to astrocytes directly triggers release of proinflammatory cytokines. To address these questions, we treated primary murine cortical astrocytes with oxidative phosphorylation (OXPHOS) inhibitors antimycin A (AA) and oligomycin A (OA) and observed the PINK1-dependent accumulation of Parkin on damaged mitochondria, leading to phospho-ubiquitination of proteins in the outer mitochondrial membrane and the recruitment of the autophagy receptor SQSTM1/p62. To identify transcriptional changes caused by mitochondrial damage and the resulting activation of mitophagic machinery, we performed bulk RNA-sequencing on astrocytes isolated from WT, PINK1-/-, or Parkin-/- mice treated with AA/OA or a vehicle control. In WT astrocytes, TNF- signaling via NF-{kappa}B was the most significantly upregulated pathway following OXPHOS inhibition. OXPHOS inhibitor treatment also stimulated p62 expression, while NF-{kappa}B inhibition prevented this upregulation. Astrocytic secretion of cytokines, including TNF-, was increased following mitochondrial damage; this secretion was dependent on NF-{kappa}B activation and occurred at levels sufficient to induce mitochondrial depolarization in hippocampal neurons. Compared to WT astrocytes, PINK1-/- astrocytes showed a significant reduction in transcriptional signatures associated with TNF- signaling following mitochondrial damage, while Parkin-/- astrocytes exhibited upregulation of both IFN-{gamma} and IFN- signaling. These findings indicate altered inflammatory responses to mitochondrial damage in the absence of functional PINK1 or Parkin. Finally, we analyzed scRNA-sequencing data from substantia nigra astrocytes harvested from human brain tissue from PD-positive or control samples. Distinct clusters comprised predominantly of PD-positive or control astrocytes emerged. Astrocytes in the PD-positive cluster were enriched for NF-{kappa}B, IFN- and IFN-{gamma} responses, consistent with the signaling observed in vitro post-OXPHOS inhibition. Together, these findings identify inflammatory signatures activated by mitochondrial damage in astrocytes, and establish this pathway as a potential contributor to neuroinflammation in PD.

SUN5 is a testis-specific SUN domain protein essential for connecting the sperm tail to the nucleus. However, until now, its precise localization, intracellular dynamics, and membrane topology during spermiogenesis have remained controversial. To address these discrepancies, we applied ultrastructure expansion microscopy (U-ExM) to systematically track SUN5 redistribution throughout spermiogenesis. This approach enabled a detailed reconstruction of SUN5 localization across developmental stages and revealed previously undescribed enrichment at the perinuclear ring (PNR) and the microtubule manchette, suggesting secondary functions at the PNR or a potential role in intra-manchette transport (IMT). Complementary immunogold labelling using the Tokuyasu method, together with biochemical assays, demonstrated that SUN5 adopts a membrane localization and topology consistent with that of classical SUN domain proteins. Quantitative measurements of the nuclear envelope architecture at the head-to-tail coupling apparatus (HTCA) further enabled us to present a refined structural model of SUN5 positioning at the head-tail junction. Overall, our findings resolve previous discrepancies in the field and provide a coherent framework for understanding SUN5 organization and its role in mammalian spermiogenesis. Summary StatementIn the presented study, we analyzed the dynamic redistribution of SUN5 during mammalian spermiogenesis and resolved its topology in developing spermatids to gain insights concerning the proteins molecular function in head-tail coupling.

African trypanosomes employ specialised mechanisms of membrane trafficking as a key strategy to persist in both the mammalian host and insect vector. Their survival and pathogenicity rely on the continuous synthesis and surface delivery of extremely abundant surface coat proteins, imposing an extraordinary biosynthetic burden on the secretory pathway. Despite this, the luminal proteome of the T. brucei endoplasmic reticulum (ER) and Golgi apparatus remains incompletely characterised. Here, we exploit TurboID proximity biotinylation, using the abundant ER chaperone BiP (Binding-immunoglobulin protein) as luminal bait to map the ER proteome in bloodstream and procyclic lifecycle stages of Trypanosoma brucei. Comparison with BiPN, a truncated secretory form of BiP that transits the Golgi, provides differential compartmental labelling, together identifying 366 (BiP) and 428 (BiPN) proximity partners respectively and encompassing established ER quality control machinery, secretory cargo, and Golgi proteins. Quantitative ranking of BiP labelling intensity identifies a cohort of candidate BiP interactors: the most strongly enriched is Tb927.5.1160, a protein sharing structural homology with the mammalian BiP nucleotide exchange inhibitor MANF (mesencephalic astrocyte-derived neurotrophic factor). Endogenous mNeonGreen tagging confirms ER localisation of TbMANF in both life cycle stages, and reciprocal manipulation of its abundance by RNAi and inducible expression produces opposing shifts in cellular sensitivity to ER stress. These data are consistent with a role in regulating BiP ATPase cycling in an organism that, unlike yeast and mammals, lacks a canonical unfolded protein response, making TbMANF the first candidate regulator of BiP activity identified in kinetoplastids. Finally, TurboID proximity labelling anchored at the inner face of the nuclear pore via NUP65 extends our endomembrane map to the inner nuclear membrane, identifying candidate proteins of this specialised ER-continuous domain. AUTHOR SUMMARYAfrican sleeping sickness is caused by Trypanosoma brucei, a parasite that survives in the mammalian bloodstream by constantly renewing its protective protein coat. To synthesise and export this surface coat, the parasite relies on two intracellular compartments, the endoplasmic reticulum (ER) and the Golgi apparatus, which function as a quality control and sorting factory for proteins entering the secretory pathway. However, the identity of the proteins that populate these compartments in blood-stage parasites, and that maintain functioning under stress conditions, has remained poorly mapped. Here, we used an enzyme-based proximity labelling strategy that identifies neighbouring proteins in live cells without disturbing their targeting signals, generating a comprehensive protein inventory of both compartments across the two main T. brucei lifecycle stages. Among the most strongly labelled proteins was Tb927.5.1160, a protein structurally related to a mammalian regulator of the master ER chaperone BiP. Reducing or increasing the abundance of Tb927.5.1160 in parasites produced opposite changes in ER stress tolerance, identifying it as a candidate modulator of ER homeostasis in a lineage that regulates protein quality control through mechanisms distinct from those operating in yeast or human cells. Together, our findings provide a new molecular resource for understanding how T. brucei sustains secretory pathway function under the biosynthetic demands of mammalian and insect host infection.

Cell and tissue-specific transcriptomic profiling of Caenorhabditis elegans is commonly achieved by fluorescence tagging or staining of targeted cell populations, often followed by fluorescence-activated cell sorting (FACS) and RNA sequencing. However, these approaches typically require separate strains for each labeled population, increasing labor and experimental variability while limiting direct comparison of multiple tissues within the same genetic background. To address this limitation and establish proof of concept, we engineered CELeidoscope, a multicolored C. elegans strain that enables spectral sorting of multiple major cell types within a single strain population. Strain construction was carried out using a high-throughput screening method that reduces the labor and plastic costs associated with transgene integration and outcrossing. Four primary tissues (body muscle, neurons, intestinal, and pharyngeal muscle cells) were tagged with spectrally distinct fluorescent proteins, allowing compatibility with viability and nucleic acid dyes. Using spectral flow cytometry, dissociated CELeidoscope cell suspensions could be sorted based on their spectral profiles, with cell recovery rates approximating the expected cell counts in whole organisms. Transcriptomic analysis of the sorted cell populations further validated the identity of the sorted populations, with recovered cells exhibiting gene expression signatures consistent with their intended cell and tissue identities. Together, these results establish CELeidoscope as a versatile tool for multiplexed cell-type isolation in C. elegans, providing a framework for tissue-specific analyses from a common strain background.

Coding mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene represent the most common cause of familial Parkinsons disease (PD), and are frequently observed in idiopathic PD. In addition, variation around the LRRK2 locus has been shown to alter PD risk by genome-wide association studies. Disease-causing mutations cluster within the catalytic core of LRRK2 - composed of GTPase (ROC) and serine-threonine kinase domains - and lead to an increase in kinase activity, resulting in hyperphosphorylation of a subset of RAB GTPases and consequent cellular toxicity. However, the interplay between LRRK2s GTPase and kinase, and with the surrounding scaffold regions has remained underexplored, with implications for the prediction of on- and off-target effects associated with kinase inhibition. To address this gap, here we dissected the contributions of kinase, GTPase and scaffold domains to LRRK2 function in murine macrophages and tissues expressing endogenous levels of GTP/GDP-binding deficient Lrrk2 T1348N. Nucleotide-free Lrrk2 is devoid of both catalytic activities but maintains the scaffold shell, leading to significant reshaping of Lrrk2 interactome and engagement in novel interactions. This altered functional state leads to impaired autophagy and accumulation of enlarged lysosomes and autophagic cargo in macrophages and kidneys. Since pharmacological inhibition of LRRK2 is under clinical evaluation, our results reveal novel gain of scaffold functions upon loss of catalytic activity that warrant careful consideration.

Plasma Membrane Calcium ATPase (PMCA) is essential for maintaining intracellular calcium homeostasis. Previously, we used constitutive PMCA downregulation in Drosophila melanogaster dopaminergic neurons as a model to increase intracellular calcium and mimic early neuronal alterations associated with Parkinsons disease. Here, we examined the mechanisms underlying the effects mediated by the conditional, adult-specific downregulation of PMCA in dopaminergic neurons in Drosophila melanogaster, both in vivo and in primary neuronal cultures. Adult-specific conditional silencing of PMCA in dopaminergic neurons reduced lifespan but to a lesser extent than the constitutive model and impaired locomotor performance. At the cellular level, PMCA-downregulated dopaminergic neurons exhibited elevated basal calcium, indicating disrupted calcium regulation. This was associated with a progressive increase in presynaptic vesicles and extracellular dopamine levels, suggesting enhanced neurotransmitter release. Notably, the synaptic active zone structure was preserved, indicating primarily functional rather than structural alterations. In primary neuronal cultures, PMCA downregulation reduced dopaminergic neuron survival and induced transient increases in neurite branching. Together, these findings show that PMCA downregulation leads to calcium dysregulation and presynaptic dysfunction without overt neurodegeneration in vivo, while promoting premature neuronal death in culture, indicating increased vulnerability and supporting a pre-degenerative state in which synaptic alterations precede neuronal loss.

The Ezrin, Radixin, and Moesin (ERM) family of proteins anchors the actin cytoskeleton to the plasma membrane for the purpose of either stabilizing or altering cell shape. In Caenorhabditis elegans, ERM-1, is essential for cell polarity, signaling, intestine development, and larval viability. Interestingly, ERM-1 proteins are produced by erm-1 mRNA transcripts that concentrate at the plasma membrane in embryos. The localization of erm-1 mRNA to the plasma membrane occurs in a 3UTR-independent, translation-dependent manner, directed by the PH-subdomain within ERM-1s N-terminal FERM domain. This has led to the model that erm-1 mRNA, its associated ribosome, and its emerging nascent peptide are all transported together to the plasma membrane as a complex. Here, we characterize the transport mechanism. Using a microscopy approach, we observed that the localizations of erm-1 mRNA and ERM-1 protein to the plasma membrane were disrupted by nocodazole treatment, illustrating a microtubule role. Furthermore, erm-1 mRNA and ERM-1 protein localized to the plasma membrane independently of myosin and dynein motors, but dependent on the kinesin bmk-1 (bmk-1), a plus-end-directed, Kinesin-5 family motor protein. Loss of bmk-1 did not reduce the total number of erm-1 mRNA molecules in the cell, arguing against a diffusion- and protection-based mechanism of mRNA localization. Together, these findings suggest that erm-1 mRNA is localized via an active transport pathway mediated by a plus-end-directed kinesin adapter. Interestingly, loss of bmk-1 led to diffuse localization of ERM-1 protein along the plasma membrane and reduced ERM-1 protein levels at the site of abscission, the midbody, and the midbody remnant. This suggests that ERM-1 local translation at the plasma membrane is critical for its proteins ultimate spatial patterning in the cell.

The microtubule and actin cytoskeletons form dynamic, interconnected networks that are critical for eukaryotic cell function. These networks govern intracellular organization, cargo transport, cell migration, and tissue morphogenesis. Microtubules and actin filaments are regulated by diverse binding proteins that control many aspects of their function. However, identifying cytoskeletal-interacting proteins has been challenging due to the transient and weak nature of many interactions and the disruption of native architecture by conventional biochemical approaches. These limitations suggest that numerous physiologically relevant cytoskeletal regulators remain undiscovered. Identifying these factors requires novel and sensitive methodologies that can capture cytoskeletal interactions under native cellular conditions. Here, we present MT-ID and Act-ID, powerful proximity-labeling tools for identifying microtubule and actin-interacting proteins, respectively. MT-ID employs the microtubule-binding domain of MAP7 (EMTB) fused to TurboID, a highly active promiscuous biotin ligase. Act-ID utilizes the actin-binding domain of ITPKA (F-tractin) similarly fused to TurboID. We validate both approaches by successfully identifying numerous known cytoskeletal regulators and discovering potentially novel interacting proteins. Functional characterization reveals that LIMCH1 is a previously unrecognized microtubule-associated protein whose depletion increases microtubule density. Additionally, we identify FBXO30 as a novel actin-interacting protein, with its loss promoting increased focal adhesion formation. MT-ID and Act-ID will be useful not only to identify cytoskeletal interacting proteins but also to define changes to the cytoskeletal interactome when cells are exposed to changing physiological conditions.