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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.

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Miro proteins are universally conserved mitochondrial calcium-binding GTPases that regulate a multitude of mitochondrial processes, including transport, clearance, and lipid trafficking. The exact role of Miro in these functions is unclear but involves binding to a variety of client proteins. How this binding is operated at the molecular level and whether and how it is important for mitochondrial health, however, remains unknown. Here, we show that known Miro interactors-namely, CENPF, Trak, and MYO19-all use a similar short motif to bind the same structural element: a highly conserved hydrophobic pocket in the first calcium-binding domain of Miro. Using these Miro-binding motifs, we identified direct interactors de novo, including MTFR1/2/1L, the lipid transporters Mdm34 and VPS13D, and the ubiquitin E3-ligase Parkin. Given the shared binding mechanism of these functionally diverse clients and its conservation across eukaryotes, we propose that Miro is a universal mitochondrial adaptor coordinating mitochondrial health.

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Membrane-bound organelles play important, frequently essential, roles in cellular metabolism in eukaryotes. Hence, cells have evolved molecular mechanisms to closely monitor organelle dynamics and maintenance. The actin cytoskeleton plays a vital role in organelle transport and positioning across all eukaryotes. Studies in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) revealed that a block in actomyosin-dependent transport affects organelle inheritance to daughter cells. Indeed, class V Myosins, Myo2, and Myo4, and many of their organelle receptors, have been identified as key factors in organelle inheritance. However, the spatiotemporal regulation of yeast organelle transport remains poorly understood. Using peroxisome inheritance as a proxy to study actomyosin-based organelle transport, we performed an automated genome-wide genetic screen in S. cerevisiae. We report that the spindle position checkpoint (SPOC) kinase Kin4 and, to a lesser extent, its paralog Frk1, regulates peroxisome transport, independent of their role in the SPOC. We show that Kin4 requires its kinase activity to function and that both Kin4 and Frk1 protect Inp2, the peroxisomal Myo2 receptor, from degradation in mother cells. In addition, vacuole inheritance is also affected in kin4/frk1-deficient cells, suggesting a common regulatory mechanism for actin-based transport for these two organelles in yeast. More broadly our findings have implications for understanding actomyosin-based transport in cells.

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In this chapter, we describe methods for reconstituting and analyzing the transport of isolated endogenous cargoes in vitro. Intracellular cargoes are transported along microtubules by teams of kinesin and dynein motors and their cargo-specific adaptor proteins. Observations from living cells show that organelles and vesicular cargoes exhibit diverse motility characteristics. Yet, our knowledge of the molecular mechanisms by which intracellular transport is regulated is not well understood. Here, we describe step-by-step protocols for the extraction of phagosomes from cells at different stages of maturation, and reconstitution of their motility along microtubules in vitro. Quantitative immunofluorescence and photobleaching techniques are also described to measure the number of motors and adaptor proteins on these isolated cargoes. In addition, we describe techniques for tracking the motility of isolated cargoes along microtubules using TIRF microscopy and quantitative force measurements using an optical trap. These methods enable us to study how the sets of motors and adaptors that drive the transport of endogenous cargoes regulate their trafficking in cells.

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Myo2, a yeast class V myosin, transports a broad range of organelles and plays important roles in various cellular processes, including cell division in budding yeast. Despite the fact that several structures of Myo2/cargo adaptor complexes have been determined, the understanding of the versatile cargo-binding modes of Myo2 is still very limited, given the large number of cargo adaptors identified for Myo2. Here, we used ColabFold, an AlphaFold2-powered and easy-to-use tool, to predict the complex structures of Myo2-GTD and its several cargo adaptors. After benchmarking the prediction strategy with three Myo2/cargo adaptor complexes that have been determined previously, we successfully predicted the atomic structures of Myo2-GTD in complex with another three cargo adaptors, Vac17, Kar9 and Pea2, which were confirmed by our biochemical characterizations. By systematically comparing the interaction details of the six complexes of Myo2 and its cargo adaptors, we summarized the cargo-binding modes on the three conserved sites of Myo2-GTD, providing an overall picture of the versatile cargo-recognition mechanisms of Myo2. In addition, our study demonstrates an efficient and effective solution to study protein-protein interactions in the future via the AlphaFold2-powered prediction.

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Intracellular membrane trafficking is a dynamic and complex cellular process. To study membrane trafficking with a high spatiotemporal resolution, we present an optogenetic method based on a blue-light inducible oligomerization of Rab GTPases, termed light-activated reversible inhibition by assembly trap of intracellular membranes (IM-LARIAT). In this chapter, we focus on the optical disruption of the dynamics and functions of previously studied intracellular membrane trafficking events, including transferrin recycling and growth cone regulation in relation to specific Rab GTPases. To aid general application, we provide a detailed description of transfection, imaging with a confocal microscope, and analysis of data.

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Axonal transport moves proteins, RNAs, and organelles between the soma and synapses to support synaptic function and activity-dependent changes in synaptic strength. This transport is impaired in several neurodegenerative disorders such as Alzheimer's disease. Thus, it is critical to understand the regulation and underlying mechanisms of the transport process. Aplysia californica provides a powerful experimental system for studying the interplay between synaptic activity and transport because its defined synaptic circuits can be built in-vitro. Advantages include precise pre- and postsynaptic manipulation, and high-resolution imaging of axonal transport. Here, we describe methodologies for the quantitative analysis of axonal transport in Aplysia sensory neurons.

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The retinal pigment epithelium (RPE) is a uniquely polarized epithelium that lies adjacent to the photoreceptor cells in the retina, and is essential for photoreceptor function and viability. Two major motile organelles present in the RPE are the melanosomes, which are important for absorbing stray light, and phagosomes that result from the phagocytosis of the distal tips of the photoreceptor cilium, known as the photoreceptor outer segment (POS). These organelles are transported along microtubules, aligned with the apical-basal axis of the RPE. Although they undergo a directional migration, the organelles exhibit bidirectional movements, indicating both kinesin and dynein motor function in their transport. Apical melanosome localization requires dynein; it has been suggested that kinesin contribution might be complex with the involvement of more than one type of kinesin. POS phagosomes undergo bidirectional movements; roles of both plus- and minus-end directed motors appear to be important in the efficient degradation of phagosomes. This function is directly related to retinal health, with defects in motor proteins, or in the association of the phagosomes with the motors, resulting in retinal degenerative pathologies.

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Several model systems have been developed to investigate mechanisms and regulation of intracellular organelle motility. The fish retinal pigment epithelial (RPE) cell represents an unusual but simple system for the study of actin-dependent organelle motility. Primary cultures of RPE dissociated from the eye are amenable to motility studies using a simple perfusion chamber and conventional phase contrast microscopy. In vivo, melanin-containing pigment granules (melanosomes) within fish RPE migrate distances up to 100 µm in response to light flux. When sheets of RPE are removed from the eye and dissociated, they attach to the substrate with apical projections extending radially from the central cell body. Melanosomes can be chemically triggered to aggregate or disperse throughout the projections. Melanosome migration in RPE apical projections is dependent on actin filaments and thus renders this model system useful for investigations of actin-dependent organelle motility.

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The Kinesin superfamily proteins (KIFs) are major molecular motors that transport diverse set of cargoes along microtubules to both the axon and dendrite of a neuron. Much of our knowledge about kinesin function is obtained from studies on axonal transport. Emerging evidence reveals how specific kinesin motor proteins carry cargoes to dendrites, including proteins, mRNAs and organelles that are crucial for synapse development and plasticity. In this review, we will summarize the major kinesin motors and their associated cargoes that have been characterized to regulate postsynaptic function in neuron. We will also discuss how specific kinesins are selectively involved in the development of excitatory and inhibitory postsynaptic compartments, their regulation by post-translational modifications (PTM), as well as their roles beyond conventional transport carrier.

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A major question in cell biology is, how are organelles and macromolecular machines moved within a cell? The delivery of cargoes to the right place at the right time within a cell is critical to cellular health. Failure to do so is often catastrophic for animal physiology and results in diseases of the gut, brain, and skin. In budding yeast, a myosin V motor, Myo2, moves cellular materials from the mother cell into the growing daughter bud. Myo2-based transport ensures that cellular contents are shared during cell division. During transport, Myo2 is often linked to its cargo via cargo-specific adaptor proteins. This simple organism thus serves as a powerful tool to study how myosin V moves cargo, such as organelles. Some critical questions include how myosin V moves along the actin cytoskeleton, or how myosin V attaches to cargo in the mother. Other critical questions include how the cargo is released from myosin V when it reaches its final destination in the bud. Here, we review the mechanisms that regulate the vacuole-specific adaptor protein, Vac17, to ensure that Myo2 delivers the vacuole to the bud and releases it at the right place and the right time. Recent studies have revealed that Vac17 is regulated by ubiquitylation and phosphorylation events that coordinate its degradation and the detachment of the vacuole from Myo2. Thus, multiple post-translational modifications tightly coordinate cargo delivery with cellular events. It is tempting to speculate that similar mechanisms regulate other cargoes and molecular motors.

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Charcot-Marie-Tooth (CMT) disease is a progressive, peripheral neuropathy and the most commonly inherited neurological disorder. Clinical manifestations of CMT mutations are typically limited to peripheral neurons, the longest cells in the body. Currently, mutations in at least 80 different genes are associated with CMT and new mutations are regularly being discovered. A large portion of the proteins mutated in axonal CMT have documented roles in mitochondrial mobility, suggesting that organelle trafficking defects may be a common underlying disease mechanism. This review will focus on the potential role of altered mitochondrial mobility in the pathogenesis of axonal CMT, highlighting the conceptional challenges and potential experimental and therapeutic opportunities presented by this "impaired mobility" model of the disease.

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Long-range movement of organelles within the cytoplasm relies on coupling to microtubule motors, a process that is often mediated by adaptor proteins. In many cases, this coupling involves organelle- or adaptor-induced activation of the microtubule motors by conformational reversal of an autoinhibited state. Herein, we show that a similar regulatory mechanism operates for an adaptor protein named SKIP (also known as PLEKHM2). SKIP binds to the small guanosine triphosphatase (GTPase) ARL8 on the lysosomal membrane to couple lysosomes to the anterograde microtubule motor kinesin-1. Structure-function analyses of SKIP reveal that the C-terminal region comprising three pleckstrin homology (PH) domains interacts with the N-terminal region comprising ARL8- and kinesin-1-binding sites. This interaction inhibits coupling of lysosomes to kinesin-1 and, consequently, lysosome movement toward the cell periphery. We also find that ARL8 does not just recruit SKIP to the lysosomal membrane but also relieves SKIP autoinhibition, promoting kinesin-1-driven, anterograde lysosome transport. Finally, our analyses show that the largely disordered middle region of SKIP mediates self-association and that this self-association enhances the interaction of SKIP with kinesin-1. These findings indicate that SKIP is not just a passive connector of lysosome-bound ARL8 to kinesin-1 but is itself subject to intra- and inter-molecular interactions that regulate its function. We anticipate that similar organelle- or GTPase-induced conformational changes could regulate the activity of other kinesin adaptors.

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Melanocytes are neuroectoderm-derived pigment-producing cells with highly polarized dendritic morphology. They protect the skin against ultraviolet radiation by providing melanin to neighbouring keratinocytes. However, the mechanisms underlying melanocyte polarization and its relevance for diseases remain mostly elusive. Numerous studies have instead revealed roles for polarity regulators in other neuroectoderm-derived lineages including different neuronal cell types. Considering the shared ontogeny and morphological similarities, these lineages may be used as reference models for the exploration of melanocyte polarity, for example, regarding dendrite formation, spine morphogenesis and polarized organelle transport. In this review, we summarize and compare the latest progress in understanding polarity regulation in neuronal cells and melanocytes and project key open questions for future work.

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Cellular function requires molecular motors to transport cargoes to their correct intracellular locations. The regulated assembly and disassembly of motor-adaptor complexes ensures that cargoes are loaded at their origin and unloaded at their destination. In Saccharomyces cerevisiae, early in the cell cycle, a portion of the vacuole is transported into the emerging bud. This transport requires a myosin V motor, Myo2, which attaches to the vacuole via Vac17, the vacuole-specific adaptor protein. Vac17 also binds to Vac8, a vacuolar membrane protein. Once the vacuole is brought to the bud cortex via the Myo2-Vac17-Vac8 complex, Vac17 is degraded and the vacuole is released from Myo2. However, mechanisms governing dissociation of the Myo2-Vac17-Vac8 complex are not well understood. Ubiquitylation of the Vac17 adaptor at the bud cortex provides spatial regulation of vacuole release. Here, we report that ubiquitylation alone is not sufficient for cargo release. We find that a parallel pathway, which initiates on the vacuole, converges with ubiquitylation to release the vacuole from Myo2. Specifically, we show that Yck3 and Vps41, independent of their known roles in homotypic fusion and protein sorting (HOPS)-mediated vesicle tethering, are required for the phosphorylation of Vac17 in its Myo2 binding domain. These phosphorylation events allow ubiquitylated Vac17 to be released from Myo2 and Vac8. Our data suggest that Vps41 is regulating the phosphorylation of Vac17 via Yck3, a casein kinase I, and likely another unknown kinase. That parallel pathways are required to release the vacuole from Myo2 suggests that multiple signals are integrated to terminate organelle inheritance.

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Rab GTPases are compartment-specific molecular switches that regulate intracellular vesicular transport in eukaryotes. GDP/GTP exchange factors (GEFs) control Rab activation, and current models propose that localised and regulated GEF activity is important in targeting Rabs to specific membranes. Here, we investigated the mechanism of GEF function using the Rab27a GEF, Rab3GEP (also known as MADD), in melanocytes as a model. We show that Rab3GEP-deficient melanocytes (melan-R3GKO) manifest partial disruption of melanosome dispersion, a read-out of Rab27a activation and targeting. Using rescue of melanosome dispersion in melan-R3GKO cells and effector pull-down approaches we show that the DENN domain of Rab3GEP (conserved among RabGEFs) is necessary, but insufficient, for its cellular function and GEF activity. Finally, using a mitochondrial re-targeting strategy, we show that Rab3GEP can target Rab27a to specific membranes in a GEF-dependent manner. We conclude that Rab3GEP facilitates the activation and targeting of Rab27a to specific membranes, but that it differs from other DENN-containing RabGEFs in requiring DENN and non-DENN elements for both of these activities and by lacking compartment-specific localisation.

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Class V myosins are actin-dependent motors, which recognize numerous cellular cargos mainly via the C-terminal globular tail domain (GTD). Myo2, a yeast class V myosin, can transport a broad range of organelles. However, little is known about the capacity of Myo2-GTD to recognize such a diverse array of cargos specifically at the molecular level. Here, we solved crystal structures of Myo2-GTD (at 1.9-3.1 Å resolutions) in complex with three cargo adaptor proteins: Smy1 (for polarization of secretory vesicles), Inp2 (for peroxisome transport), and Mmr1 (for mitochondria transport). The structures of Smy1- and Inp2-bound Myo2-GTD, along with site-directed mutagenesis experiments, revealed a binding site in subdomain-I having a hydrophobic groove with high flexibility enabling Myo2-GTD to accommodate different protein sequences. The Myo2-GTD-Mmr1 complex structure confirmed and complemented a previously identified mitochondrion/vacuole-specific binding region. Moreover, differences between the conformations and locations of cargo-binding sites identified here for Myo2 and those reported for mammalian MyoVA (MyoVA) suggest that class V myosins potentially have co-evolved with their specific cargos. Our structural and biochemical analysis not only uncovers a molecular mechanism that explains the diverse cargo recognition by Myo2-GTD, but also provides structural information useful for future functional studies of class V myosins in cargo transport.

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Multiple transporters and channels mediate cation transport across the plasma membrane and tonoplast to regulate ionic homeostasis in plant cells. However, much less is known about the molecular function of transporters that facilitate cation transport in other organelles such as Golgi. We report here that Arabidopsis KEA4, KEA5, and KEA6, members of cation/proton antiporters-2 (CPA2) superfamily were colocalized with the known Golgi marker, SYP32-mCherry. Although single kea4,5,6 mutants showed similar phenotype as the wild type under various conditions, kea4/5/6 triple mutants showed hypersensitivity to low pH, high K+ , and high Na+ and displayed growth defects in darkness, suggesting that these three KEA-type transporters function redundantly in controlling etiolated seedling growth and ion homeostasis. Detailed analysis indicated that the kea4/5/6 triple mutant exhibited cell wall biosynthesis defect during the rapid etiolated seedling growth and under high K+ /Na+ condition. The cell wall-derived pectin homogalacturonan (GalA)3 partially suppressed the growth defects and ionic toxicity in the kea4/5/6 triple mutants when grown in the dark but not in the light conditions. Together, these data support the hypothesis that the Golgi-localized KEAs play key roles in the maintenance of ionic and pH homeostasis, thereby facilitating Golgi function in cell wall biosynthesis during rapid etiolated seedling growth and in coping with high K+ /Na+ stress.

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The posttranslational modification (PTM) of tubulin subunits is important for the physiological functions of the microtubule (MT) cytoskeleton. Although major advances have been made in the identification of enzymes carrying out MT-PTMs, little knowledge is available on how intercellular signaling molecules and their associated pathways regulate MT-PTM-dependent processes inside signal-receiving cells. Here we show that Hedgehog (Hh) signaling, a paradigmatic intercellular signaling system, affects the MT acetylation state in mammalian cells. Mechanistically, Hh pathway activity increases the levels of the MT-associated DYRK1B kinase, resulting in the inhibition of GSK3ß through phosphorylation of Serine 9 and the subsequent suppression of HDAC6 enzyme activity. Since HDAC6 represents a major tubulin deacetylase, its inhibition increases the levels of acetylated MTs. Through the activation of DYRK1B, Hh signaling facilitates MT-dependent processes such as intracellular mitochondrial transport, mesenchymal cell polarization or directed cell migration. Taken together, we provide evidence that intercellular communication through Hh signals can regulate the MT cytoskeleton and contribute to MT-dependent processes by affecting the level of tubulin acetylation.

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Advances in cell biology have been largely driven by pioneering work in model systems, the majority of which are from one major eukaryotic lineage, the opisthokonts. However, with the explosion of genomic information in many lineages, it has become clear that eukaryotes have incredible diversity in many cellular systems, including the cytoskeleton. By identifying model systems in diverse lineages, it may be possible to begin to understand the evolutionary origins of the eukaryotic cytoskeleton. Within the plant lineage, cell biological studies in the model moss, Physcomitrella patens, have over the past decade provided key insights into how the cytoskeleton drives cell and tissue morphology. Here, we review P. patens attributes that make it such a rich resource for cytoskeletal cell biological inquiry and highlight recent key findings with regard to intracellular transport, microtubule-actin interactions, and gene discovery that promises for many years to provide new cytoskeletal players.