Cargo Trafficking Research Articles

Small ARF-like 2 GTPase TITAN 5 is linked with the dynamic regulation of IRON-REGULATED TRANSPORTER 1.

Iron acquisition is crucial for plants. The abundance of IRON-REGULATED TRANSPORTER 1 (IRT1) is controlled through endomembrane trafficking, a process that requires small ARF-like GTPases. Only few components are known that are involved in the vesicular trafficking of specific cargo. Here, we report that the ARF-like GTPase TITAN 5 (TTN5) interacts with the large cytoplasmic variable region and protein-regulatory platform of IRT1. Heterozygous ttn5-1 plants can display a reduced activity of root iron reductase. This activity is needed for iron uptake via IRT1. Fluorescent fusion proteins of TTN5 and IRT1 colocalize at places, where IRT1 sorting and cycling between the plasma membrane and the vacuole are coordinated. TTN5 can also interact with peripheral membrane proteins that are components of the IRT1 regulation machinery, like the trafficking factor SNX1, the C2 domain protein EHB1 and the SEC14-GOLD protein PATL2. Hence, the link between iron acquisition and vesicular trafficking involving a small GTPase of the ARF family opens up the possibility to study the involvement of TTN5 in nutritional cell biology in the endomembrane system.

Subtleties in Clathrin heavy chain binding boxes provide selectivity among adaptor proteins of budding yeast

Clathrin forms a triskelion, or three-legged, network that regulates cellular processes by facilitating cargo internalization and trafficking in eukaryotes. Its N-terminal domain is crucial for interacting with adaptor proteins, which link clathrin to the membrane and engage with specific cargo. The N-terminal domain contains up to four adaptor-binding sites, though their role in preferential occupancy by adaptor proteins remains unclear. In this study, we examine the binding hierarchy of adaptors for clathrin, using integrative biophysical and structural approaches, along with in vivo functional experiments. We find that yeast epsin Ent5 has the highest affinity for clathrin, highlighting its key role in cellular trafficking. Epsins Ent1 and Ent2, crucial for endocytosis but thought to have redundant functions, show distinct binding patterns. Ent1 exhibits stronger interactions with clathrin than Ent2, suggesting a functional divergence toward actin binding. These results offer molecular insights into adaptor protein selectivity, suggesting they competitively bind clathrin while also targeting three different clathrin sites.

Screens for mutants defective in UapA trafficking highlight the importance of ER-exit as a primary control point in transporter biogenesis

Most transmembrane membrane proteins are thought to traffic to the plasma membrane (PM) via the conventional secretory pathway through sorting from the Golgi. However, our recent work has shown that in the filamentous fungus Aspergillus nidulans several nutrient transporters and other major membrane proteins traffic to the PM via Golgi-bypass and independently of known post-Golgi secretory mechanisms. Here in an effort to dissect the molecular mechanism underlying membrane cargo trafficking via Golgi-bypass we design and use unbiased genetic screens, based on the UapA uric acid-xanthine transporter, which allowed the isolation of mutants defective in UapA translocation to the plasma membrane. Analyses of these mutants highlight the importance of ER-exit as the primary control point in transporter trafficking via Golgi-bypass. Most mutants isolated concerned mutations within the uapA gene, albeit we also obtained uapA extragenetic mutants affecting secretion and growth pleiotropically or leading or apparent activation of an efflux transporter related to purine-detoxification. Our work paves the way to use genetic approaches targeting specifically trafficking mutations affecting Golgi-bypass.

Targeting Tetraspanins at Cell Interfaces: Functional Modulation and Exosome-Based Drug Delivery for Precise Disease Treatment.

Tetraspanins are key players in various physiological and pathological processes, including malignancy, immune response, fertilization, and infectious disease. Affinity ligands targeting the interactions between tetraspanins and partner proteins are promising for modulating downstream signaling pathways, thus emerging as attractive candidates for interfering related biological functions. Due to the involvement in vesicle biogenesis and cargo trafficking, tetraspanins are also regarded as exosome markers, and become molecular targets for drug loading and delivery. Given the rapid development in these areas, this minireview focuses on recent advances in design and engineering of affinity binders toward tetraspanins including CD63, CD81, and CD9. Their mechanism of actions in modulating protein interactions at cell interfaces and treatment of malignant diseases are discussed. Strategies for constructing exosome-based drug delivery platforms are also reviewed, with emphasis on the important roles of tetraspanins and the affinity ligands. Finally, challenges and future development of tetraspanin-targeting therapy and exosomal drug delivery platforms are also discussed.

Impaired hippocampal plasticity associated with loss of recycling endosomal SLC9A6/NHE6 is ameliorated by the TrkB agonist 7,8-dihydroxyflavone

Proper maintenance of intracellular vesicular pH is essential for cargo trafficking during synaptic function and plasticity. Mutations in the SLC9A6 gene encoding the recycling endosomal pH regulator (Na+, K+)/H+ exchanger isoform 6 (NHE6) are causal for Christianson syndrome (CS), a severe form of X-linked intellectual disability. NHE6 expression is also downregulated in other neurodevelopmental and neurodegenerative disorders, such as autism spectrum disorder and Alzheimer's disease, suggesting its dysfunction could contribute more broadly to the pathophysiology of other neurological conditions. To understand how ablation of NHE6 function leads to severe learning impairments, we assessed synaptic structure, function, and cellular mechanisms of learning in a novel line of Nhe6 knockout (KO) mice expressing a plasma membrane-tethered green fluorescent protein within hippocampal neurons. We uncovered significant reductions in dendritic spines density, AMPA receptor (AMPAR) expression, and AMPAR-mediated neurotransmission in CA1 pyramidal neurons. The neurons also failed to undergo functional and structural enhancement during long-term potentiation (LTP). Significantly, the selective TrkB agonist 7,8-dihydroxyflavone restored spine density as well as functional and structural LTP in KO neurons. TrkB activation thus may act as a potential clinical intervention to ameliorate cognitive deficits in CS and other neurodegenerative disorders.

The Role of Cornichons in the Biogenesis and Functioning of Monovalent-Cation Transport Systems.

Monovalent-cation homeostasis, crucial for all living cells, is ensured by the activity of various types of ion transport systems located either in the plasma membrane or in the membranes of organelles. A key prerequisite for the functioning of ion-transporting proteins is their proper trafficking to the target membrane. The cornichon family of COPII cargo receptors is highly conserved in eukaryotic cells. By simultaneously binding their cargoes and a COPII-coat subunit, cornichons promote the incorporation of cargo proteins into the COPII vesicles and, consequently, the efficient trafficking of cargoes via the secretory pathway. In this review, we summarize current knowledge about cornichon proteins (CNIH/Erv14), with an emphasis on yeast and mammalian cornichons and their role in monovalent-cation homeostasis. Saccharomyces cerevisiae cornichon Erv14 serves as a cargo receptor of a large portion of plasma-membrane proteins, including several monovalent-cation transporters. By promoting the proper targeting of at least three housekeeping ion transport systems, Na+, K+/H+ antiporter Nha1, K+ importer Trk1 and K+ channel Tok1, Erv14 appears to play a complex role in the maintenance of alkali-metal-cation homeostasis. Despite their connection to serious human diseases, the repertoire of identified cargoes of mammalian cornichons is much more limited. The majority of current information is about the structure and functioning of CNIH2 and CNIH3 as auxiliary subunits of AMPAR multi-protein complexes. Based on their unique properties and easy genetic manipulation, we propose yeast cells to be a useful tool for uncovering a broader spectrum of human cornichons´ cargoes.

Neurons Specialize in Presynaptic Autophagy: A Perspective to Ameliorate Neurodegeneration.

The efficient and prolonged neurotransmission is reliant on the coordinated action of numerous synaptic proteins in the presynaptic compartment that remodels synaptic vesicles for neurotransmitter packaging and facilitates their exocytosis. Once a cycle of neurotransmission is completed, membranes and associated proteins are endocytosed into the cytoplasm for recycling or degradation. Both exocytosis and endocytosis are closely regulated in a timely and spatially constrained manner. Recent research demonstrated the impact of dysfunctional synaptic vesicle retrieval in causing retrograde degeneration of midbrain neurons and has highlighted the importance of such endocytic proteins, including auxilin, synaptojanin1 (SJ1), and endophilin A (EndoA) in neurodegenerative diseases. Additionally, the role of other associated proteins, including leucine-rich repeat kinase 2 (LRRK2), adaptor proteins, and retromer proteins, is being investigated for their roles in regulating synaptic vesicle recycling. Research suggests that the degradation of defective vesicles via presynaptic autophagy, followed by their recycling, not only revitalizes them in the active zone but also contributes to strengthening synaptic plasticity. The presynaptic autophagy rejuvenating terminals and maintaining neuroplasticity is unique in autophagosome formation. It involves several synaptic proteins to support autophagosome construction in tiny compartments and their retrograde trafficking toward the cell bodies. Despite having a comprehensive understanding of ATG proteins in autophagy, we still lack a framework to explain how autophagy is triggered and potentiated in compact presynaptic compartments. Here, we reviewed synaptic proteins' involvement in forming presynaptic autophagosomes and in retrograde trafficking of terminal cargos. The review also discusses the status of endocytic proteins and endocytosis-regulating proteins in neurodegenerative diseases and strategies to combat neurodegeneration.

TFG regulates inner COPII coat recruitment to facilitate anterograde secretory protein transport.

Coat protein complex II (COPII) governs the initial steps of biosynthetic secretory protein transport from the endoplasmic reticulum (ER), facilitating the movement of a wide variety of cargoes. Here, we demonstrate that Trk-fused gene (TFG) regulates the rate at which inner COPII coat proteins are concentrated at ER subdomains. Specifically, in cells lacking TFG, the GTPase-activating protein (GAP) Sec23 accumulates more rapidly at budding sites on the ER as compared with control cells, potentially altering the normal timing of GTP hydrolysis on Sar1. Under these conditions, anterograde trafficking of several secretory cargoes is delayed, irrespective of their predicted size. We propose that TFG controls the local, freely available pool of Sec23 during COPII coat formation and limits its capacity to prematurely destabilize COPII complexes on the ER. This function of TFG enables it to act akin to a rheostat, promoting the ordered recruitment of Sec23, which is critical for efficient secretory cargo export.

SNX32 Regulates Sorting and Trafficking of Activated EGFR to the Lysosomal Degradation Pathway.

SNX32 is a member of the evolutionarily conserved Phox (PX) homology domain- and Bin/Amphiphysin/Rvs (BAR) domain-containing sorting nexin (SNX-BAR) family of proteins, which play important roles in sorting and membrane trafficking of endosomal cargoes. Although SNX32 shares the highest amino acid sequence homology with SNX6, and has been believed to function redundantly with SNX5 and SNX6 in retrieval of the cation-independent mannose-6-phosphate receptor (CI-MPR) from endosomes to the trans-Golgi network (TGN), its role(s) in intracellular protein trafficking remains largely unexplored. Here, we report that it functions in parallel with SNX1 in mediating epidermal growth factor (EGF)-stimulated postendocytic trafficking of the epidermal growth factor receptor (EGFR). Moreover, SNX32 interacts directly with EGFR, and recruits SNX5 to promote sorting of EGF-EGFR into multivesicular bodies (MVBs) for lysosomal degradation. Thus, SNX32 functions distinctively from other SNX-BAR proteins to mediate signaling-coupled endolysosomal trafficking of EGFR.

A force-sensitive mutation reveals a non-canonical role for dynein in anaphase progression.

The diverse roles of the dynein motor in shaping microtubule networks and cargo transport complicate in vivo analysis of its functions significantly. To address this issue, we have generated a series of missense mutations in Drosophila Dynein heavy chain. We show that mutations associated with human neurological disease cause a range of defects, including impaired cargo trafficking in neurons. We also describe a novel microtubule-binding domain mutation that specifically blocks the metaphase-anaphase transition during mitosis in the embryo. This effect is independent from dynein's canonical role in silencing the spindle assembly checkpoint. Optical trapping of purified dynein complexes reveals that this mutation only compromises motor performance under load, a finding rationalized by the results of all-atom molecular dynamics simulations. We propose that dynein has a novel function in anaphase progression that depends on it operating in a specific load regime. More broadly, our work illustrates how in vivo functions of motors can be dissected by manipulating their mechanical properties.

CCSer2 gates dynein activity at the cell periphery.

Cytoplasmic dynein-1 (dynein) is a microtubule-associated, minus end-directed motor that traffics hundreds of different cargos. Dynein must discriminate between cargos and traffic them at the appropriate time from the correct cellular region. How dynein's trafficking activity is regulated in time or cellular space remains poorly understood. Here, we identify CCSer2 as the first known protein to gate dynein activity in the spatial dimension. CCSer2 promotes the migration of developing zebrafish primordium cells and of cultured human cells by facilitating the trafficking of cargos that are acted on by cortically localized dynein. CCSer2 inhibits the interaction between dynein and its regulator Ndel1 exclusively at the cell periphery, resulting in localized dynein activation. Our findings suggest that the spatial specificity of dynein is achieved by the localization of proteins that disinhibit Ndel1. We propose that CCSer2 defines a broader class of proteins that activate dynein in distinct microenvironments via Ndel1 inhibition.

Behavioral and genetic correlates of heterogeneity in learning performance in individual honeybees, Apis mellifera.

Learning an olfactory discrimination task leads to heterogeneous results in honeybees with some bees performing very well and others at low rates. Here we investigated this behavioral heterogeneity and asked whether it was associated with particular gene expression patterns in the bee's brain. Bees were individually conditioned using a sequential conditioning protocol involving several phases of olfactory learning and retention tests. A cumulative score was used to differentiate the tested bees into high and low performers. The rate of CS+ odor learning was found to correlate most strongly with a cumulative performance score extracted from all learning and retention tests. Microarray analysis of gene expression in the mushroom body area of the brains of these bees identified a number of differentially expressed genes between high and low performers. These genes are associated with diverse biological functions, such as neurotransmission, memory formation, cargo trafficking and development.

γ-secretase facilitates retromer-mediated retrograde transport.

The retromer complex mediates retrograde transport of protein cargos from endosomes to the trans-Golgi network (TGN). γ-secretase is a multisubunit protease that cleaves the transmembrane domain of its target proteins. Mutations in genes encoding subunits of retromer or γ-secretase can cause familial Alzheimer disease (AD) and other degenerative neurological diseases. It has been reported that retromer interacts with γ-secretase, but the consequences of this interaction are not known. Here, we report that retromer-mediated retrograde protein trafficking in cultured human epithelial cells is impaired by inhibition of γ-secretase activity or by genetic elimination of γ-secretase. γ-secretase inhibitor XXI and knockout of PS1, the catalytic subunit of γ-secretase, inhibit endosome to TGN trafficking of retromer-dependent retrograde cargos, divalent metal transporter 1 isoform II (DMT1-II), cation-independent mannose-6-phosphate receptor (CIMPR), and shiga toxin. Trafficking of retromer-independent cargos, such as cholera toxin and a CIMPR mutant that does not bind to retromer was not affected by γ-secretase inhibition. XXI treatment and PS1 KO inhibit interaction of γ-secretase with retromer but do not inhibit the association of cargo with retromer or with γ-secretase in intact cells. Similarly, these treatments do not affect the level of Rab7-GTP, which regulates retromer-cargo interaction. These results suggest that the γ-secretase-retromer interaction facilitates retromer-mediated retrograde trafficking.

Label-Free Interference Imaging of Intracellular Trafficking.

Intracellular cargo trafficking is a highly regulated process responsible for transporting vital cellular components to their designated destinations. This intricate journey has been a central focus of cellular biology for many years. Early investigations leaned heavily on biochemical and genetic approaches, offering valuable insight into molecular mechanisms of cellular trafficking. However, while informative, these methods lack the capacity to capture the dynamic nature of intracellular trafficking. The advent of fluorescent protein tagging techniques transformed our ability to monitor the complete lifecycle of intracellular cargos, advancing our understanding. Yet, a central question remains: How do these cargos manage to navigate through traffic challenges, such as congestion, within the crowded cellular environment? Fluorescence-based imaging, though valuable, has inherent limitations when it comes to addressing the aforementioned question. It is prone to photobleaching, making long-term live-cell imaging challenging. Furthermore, they render unlabeled cellular constituents invisible, thereby missing critical environmental information. Notably, the unlabeled majority likely exerts a significant influence on the observed behavior of labeled molecules. These considerations underscore the necessity of developing complementary label-free imaging methods to overcome the limitations of fluorescence imaging or to integrate them synergistically.In this Account, we outline how label-free interference-based imaging has the potential to revolutionize the study of intracellular traffic by offering unprecedented levels of detail. We begin with a brief introduction to our previous findings in live-cell research enabled by interferometric scattering (iSCAT) microscopy, showcasing its aptitude and adeptness in elucidating intricate nanoscale intracellular structures. As we delved deeper into our exploration, we succeeded in the label-free visualization of the entire lifespan of nanoscale protein complexes known as nascent adhesions (NAs) and the dynamic events associated with adhesions within living cells. Our continuous efforts have led to the development of Dynamic Scattering-particle Localization Interference Microscopy (DySLIM), a generalized concept of cargo-localization iSCAT (CL-iSCAT). This label-free, high-speed imaging method, armed with iSCAT detection sensitivity, empowers us to capture quantitative and biophysical insights into cargo transport, providing a realistic view of the intricate nanoscale logistics occurring within living cells. Our in vivo studies demonstrate that intracellular cargos regularly contend with substantial traffic within the crowded cellular environment. Simultaneously, they employ inherent strategies for efficient cargo transport, such as collective migration and hitchhiking, to enhance overall transport rates─intriguingly paralleling the principle and practice of urban traffic management. We also highlight the synergistic benefits of combining DySLIM with chemical-selective fluorescent methods. This Account concludes with a "Conclusions and Outlook" section, outlining promising directions for future research and developments, with a particular emphasis on the functional application of iSCAT live-cell imaging. We aim to inspire further investigation into the efficient transport strategies employed by cells to surmount transportation challenges, shedding light on their significance in cellular phenomena.

More than meets the eye: knowns and unknowns of the trafficking of small secreted proteins in Arabidopsis.

Small proteins represent a significant portion of the cargo transported through plant secretory pathways, playing crucial roles in developmental processes, fertilization, and responses to environmental stresses. Despite the importance of small secreted proteins, substantial knowledge gaps persist regarding the regulatory mechanisms governing their trafficking along the secretory pathway, and their ultimate localization or destination. To address these gaps, we conducted a comprehensive literature review, focusing particularly on trafficking and localization of Arabidopsis small secreted proteins with potential biochemical and/or signaling roles in the extracellular space, typically those within the size range of 101-200 amino acids. Our investigation reveals that while at least six members of the 21 mentioned families have a confirmed extracellular localization, eight exhibit intracellular localization, including cytoplasmic, nuclear, and chloroplastic locations, despite the presence of N-terminal signal peptides. Further investigation into the trafficking and secretion mechanisms of small protein cargo could not only deepen our understanding of plant cell biology and physiology but also provide a foundation for genetic manipulation strategies leading to more efficient plant cultivation.

Mapping the global interactome of the ARF family reveals spatial organization in cellular signaling pathways.

The ADP-ribosylation factors (ARFs) and ARF-like (ARL) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we used proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified ∼3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely, SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.

Abstract 154: Hierarchical Redundancy And Contextual Roles Of Vesicle-associated Membrane Proteins (VAMPs) In Platelet Function

Background: Platelet granule secretion, driven by the SNARE complex involving Vesicle-Associated Membrane Proteins (VAMPs) and t-SNAREs, is essential for vascular integrity. We dissected individual and combinatorial contributions of all four platelet VAMPs to granule secretion, hemostasis, and intracellular cargo trafficking. Research Questions: 1. Are specific VAMPs critical for granule fusion, or does total VAMP quantity (mass action) dictate secretion? 2. How do differential VAMP deficiencies impact hemostasis in arterial and venous microenvironments? 3. Do VAMPs influence platelet cargo loading and gene expression? Goals: Investigate individual and combinatorial roles of VAMPs in platelet biology and their context dependent contributions to hemostasis. Approach: We generated V7 -/- 8 -/- and V2 Δ 3 Δ 7 -/- 8 -/- mice, assessing granule secretion (dense, alpha, lysosomal) via ATP, PF4/P-selectin, and β-hexosaminidase/LAMP1 release assays, respectively. Hemostasis in arterial and venous models was evaluated with tail bleeding, FeCl3, and venous puncture injury assays. Cargo levels were analyzed using proteomic arrays, and platelet RNA sequencing identified transcriptional changes. Results: Global V7/V8 deletion modestly reduced secretion compared to isolated V8 deficiency. Surprisingly, while platelet-specific V2 Δ 3 Δ 7 -/- 8 -/- exhibited the most dramatic decrease, complete abolition was absent (p<0.01). Alpha and lysosomal secretion were more affected than densegranule release. Both knockout models bled profusely and failed to form stable arterial thrombi (p<0.01). Notably, only V2 Δ 3 Δ 7 -/- 8 -/- mice displayed significantly prolonged venous bleeding (p<0.01). RNA sequencing revealed >800 upregulated and >500 downregulated transcripts in V7 - /- 8 -/- and V2 Δ 3 Δ 8 -/- platelets, suggesting shared transcriptional control by V7 and secondary VAMPs (V2 and V3). Conclusions: VAMP functions exhibit hierarchical redundancy, with V8 presence minimally impacting dense granule secretion and arterial hemostasis. Alpha granule release is more sensitive to VAMP loss. The requirements for platelet granule secretion for hemostasis are context dependent. The deletion of VAMPs disrupts platelet cargo trafficking and transcription.

Loss of tumor suppressor TMEM127 drives RET-mediated transformation through disrupted membrane dynamics

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

Loss of tumor suppressor TMEM127 drives RET-mediated transformation through disrupted membrane dynamics.

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

The types and numbers of kinesins and dyneins transporting endocytic cargoes modulate their motility and response to tau

Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different stages of maturation and reconstituted their motility along microtubules in vitro. We asked how the sets of motors transporting a cargo determine its motility and response to the microtubule-associated protein tau. Here, we find that phagosomes move in both directions along microtubules, but the directional bias changes during maturation. Early phagosomes exhibit retrograde-biased transport while late phagosomes are directionally unbiased. Correspondingly, early and late phagosomes are bound by different numbers and combinations of kinesins -1, -2, -3, and dynein. Tau stabilizes microtubules and directs transport within neurons. While single-molecule studies show that tau differentially regulates the motility of kinesins and dynein in vitro, less is known about its role in modulating the trafficking of endogenous cargoes transported by their native teams of motors. Previous studies showed that tau preferentially inhibits kinesin motors, which biases late phagosome transport towards the microtubule minus-end. Here, we show that tau strongly inhibits long-range, dynein-mediated motility of early phagosomes. Tau reduces forces generated by teams of dynein motors on early phagosomes and accelerates dynein unbinding under load. Thus, cargoes differentially respond to tau, where dynein-complexes on early phagosomes are more sensitive to tau inhibition than those on late phagosomes. Mathematical modeling further explains how small changes in the number of kinesins and dynein on cargoes impact the net directionality but also that cargoes with different sets of motors respond differently to tau.