Organelle Transport Research Articles

Actin–myosin XI: an intracellular control network in plants

Actin is one of the three major cytoskeletal components in eukaryotic cells. Myosin XI is an actin-based motor protein in plant cells. Organelles are attached to myosin XI and translocated along the actin filaments. This dynamic actin–myosin XI system plays a major role in subcellular organelle transport and cytoplasmic streaming. Previous studies have revealed that myosin-driven transport and the actin cytoskeleton play essential roles in plant cell growth. Recent data have indicated that the actin–myosin XI cytoskeleton is essential for not only cell growth but also reproductive processes and responses to the environment. In this review, we have summarized previous reports regarding the role of the actin–myosin XI cytoskeleton in cytoplasmic streaming and plant development and recent advances in the understanding of the functions of actin–myosin XI cytoskeleton in Arabidopsis thaliana.

Multiple Roles, Multiple Adaptors: Dynein During Cell Cycle.

Dynein is an essential protein complex present in most eukaryotes that regulate biological processes ranging from ciliary beating, intracellular transport, to cell division. Elucidating the detailed mechanism of dynein function has been a challenging task owing to its large molecular weight and high complexity of the motor. With the advent of technologies in the last two decades, studies have uncovered a wealth of information about the structural, biochemical, and cell biological roles of this motor protein. Cytoplasmic dynein associates with dynactin through adaptor proteins to mediate retrograde transport of vesicles, mRNA, proteins, and organelles on the microtubule tracts. In a mitotic cell, dynein has multiple localizations, such as at the nuclear envelope, kinetochores, mitotic spindle and spindle poles, and cell cortex. In line with this, dynein regulates multiple events during the cell cycle, such as centrosome separation, nuclear envelope breakdown, spindle assembly checkpoint inactivation, chromosome segregation, and spindle positioning. Here, we provide an overview of dynein structure and function with focus on the roles played by this motor during different stages of the cell cycle. Further, we review in detail the role of dynactin and dynein adaptors that regulate both recruitment and activity of dynein during the cell cycle.

Axonal transport in Neurodegenerative diseases

The transport of organelles through an axon into the cell is very important for the maintenance and function of a neuron. Antherograde axonal transport plays a role in the supply of distal synapse and mitochondrial proteins and lipids for local energy requirements, and is involved in intracellular transport of retrograde transport, misfolded and aggregated proteins from the axon and distal trophic sites to the somatic. Axonal transport can be affected by changes to various components of the transport mechanism. Here we review the available information about axonal transport defects that may contribute to the pathogenesis of certain neurodegenerative diseases.

Protein Gradients on the Nucleoid Position the Carbon-Fixing Organelles of Cyanobacteria

Carboxysomes are protein-based bacterial organelles that encapsulate a key enzyme of the Calvin-Benson-Bassham cycle. Previous work has implicated a ParA-like protein (hereafter McdA) as important for spatially organizing carboxysomes along the longitudinal axis of the model cyanobacterium Synechococcus elongatus PCC 7942. Yet, how self-organization of McdA emerges and contributes to carboxysome positioning is unknown. Here, we show that a small protein, termed McdB, localizes to carboxysomes through interactions with carboxysome shell proteins to drive emergent oscillatory patterning of McdA on the nucleoid. Our results demonstrate that McdB directly interacts to stimulate McdA ATPase activity, and indicate that carboxysome-dependent McdA depletion zone formation on the nucleoid is required for directed motion of carboxysomes towards increased concentrations of McdA. We propose that McdA and McdB are a new class of self-organizing proteins that follow a Brownian-ratchet mechanism, challenging the cytoskeletal model of organelle transport, for equidistant positioning of carboxysomes in cyanobacteria. These results have broader implications for understanding spatial organization of protein mega-complexes and organelles in bacteria more broadly.

Mechanisms of copper and zinc homeostasis in pathogenic black fungi

Black fungi comprise a diverse group of melanized microorganisms, many of which are able to infect humans. One of the recognized diseases that arise with black fungi infection is chromoblastomycosis, a neglected implantation mycosis. Considering their ecology, black fungi may face conditions with distinct metal availability. Zinc and copper are essential transition metals, which become toxic in excess. During the interaction with host, fungi may face either metal deprivation or poisoning. Here we report an in silico analysis of four black fungi genomes concerning zinc and copper homeostasis. Overall, these organisms share apparatus of metal uptake, storage and detoxification with other pathogenic and non-pathogenic fungi. Genes coding plasma membrane and organelle transporters, as well as metal binding proteins were identified. Althought putatives zinc and copper responsive transcription factors have been found in the analyzed genomes, remarkable structural differences were perceived when compared to the already characterized regulators. Black fungi may harbor unique features concerning the regulation of zinc and copper homeostasis, which is probably a result of the niches they can inhabit. The data provided here add knowlegde to a still unexplored aspect of black fungi biology that may be useful in the understanding of their pathogenicity.

A small-molecule activator of kinesin-1 drives remodeling of the microtubule network

The microtubule motor kinesin-1 interacts via its cargo-binding domain with both microtubules and organelles, and hence plays an important role in controlling organelle transport and microtubule dynamics. In the absence of cargo, kinesin-1 is found in an autoinhibited conformation. The molecular basis of how cargo engagement affects the balance between kinesin-1's active and inactive conformations and roles in microtubule dynamics and organelle transport is not well understood. Here we describe the discovery of kinesore, a small molecule that in vitro inhibits kinesin-1 interactions with short linear peptide motifs found in organelle-specific cargo adaptors, yet activates kinesin-1's function of controlling microtubule dynamics in cells, demonstrating that these functions are mechanistically coupled. We establish a proof-of-concept that a microtubule motor-cargo interface and associated autoregulatory mechanism can be manipulated using a small molecule, and define a target for the modulation of microtubule dynamics.

Pleiotropic neuropathological and biochemical alterations associated with Myo5a mutation in a rat Model

In this study, we analyze the neuropathological and biochemical alterations involved in the pathogenesis of a neurodegenerative/movement disorder during different developmental stages in juvenile rats with a mutant Myosin5a (Myo5a). In mutant rats, a spontaneous autosomal recessive mutation characterized by the absence of Myo5a protein expression in the brain is associated with a syndrome of locomotor dysfunction, altered coat color, and neuroendocrine abnormalities. Myo5a encodes a myosin motor protein required for transport and proper distribution of subcellular organelles in somatodendritic processes in neurons. Here we report marked hyperphosphorylation of alpha-synuclein and tau, as well as region-specific buildup of the autotoxic dopamine metabolite, 3,4-dihydroxyphenyl-acetaldehyde (DOPAL), related to decreased aldehyde dehydrogenases activity and neurodegeneration in mutant rats. Alpha-synuclein accumulation in mitochondria of dopaminergic neurons is associated with impaired enzymatic respiratory complex I and IV activity. The behavioral and biochemical lesions progress after 15 days postnatal, and by 30–40 days the animals must be euthanized because of neurological impairment. Based on the obtained results, we propose a pleiotropic pathogenesis that links the Myo5a gene mutation to deficient neuronal development and progressive neurodegeneration. This potential model of a neurodevelopmental disorder with neurodegeneration and motor deficits may provide further insight into molecular motors and their associated proteins responsible for altered neurogenesis and neuronal disease pathogenesis.

Cover Picture

AbstractThe review by Kaczmarek et al. describes new functions of the Arf1 small G protein and its activator GBF1, beyond their well‐known role in formation of retrograde transport vesicles between the endoplasmic reticulum and the Golgi apparatus. These new functions include roles in lipid droplet metabolism, clathrin‐independent endocytosis, mitochondrial dynamics and transport of organelles along microtubules.

Low-dimensional manifold of actin polymerization dynamics

Actin filaments are critical components of the eukaryotic cytoskeleton, playing important roles in a number of cellular functions, such as cell migration, organelle transport, and mechanosensation. They are helical polymers with a well-defined polarity, composed of globular subunits that bind nucleotides in one of three hydrolysis states (ATP, ADP-Pi, or ADP). Mean-field models of the dynamics of actin polymerization have succeeded in, among other things, determining the nucleotide profile of an average filament and resolving the mechanisms of accessory proteins. However, these models require numerical solution of a high-dimensional system of nonlinear ordinary differential equations. By truncating a set of recursion equations, the Brooks–Carlsson (BC) model reduces dimensionality to 11, but it still remains nonlinear and does not admit an analytical solution, hence, significantly hindering understanding of its resulting dynamics. In this work, by taking advantage of the fast timescales of the hydrolysis states of the filament tips, we propose two model reduction schemes: the quasi steady-state approximation model is five-dimensional and nonlinear, whereas the constant tip (CT) model is five-dimensional and linear, resulting from the approximation that the tip states are not dynamic variables. We provide an exact solution of the CT model and use it to shed light on the dynamical behaviors of the full BC model, highlighting the relative ordering of the timescales of various collective processes, and explaining some unusual dependence of the steady-state behavior on initial conditions.

Cell scientist to watch – Lukas Kapitein

ABSTRACT Lukas Kapitein studied physics at the VU University in Amsterdam and graduated with a master's degree in experimental physics. He went on to do his PhD in the research groups of Christoph Schmidt and Erwin Peterman on the motility of mitotic kinesins, and in 2007, he won the biannual best thesis award from the Dutch society for Biophysics and Biomedical Technology. Lukas then joined the laboratory of Casper Hoogenraad at the Erasmus Medical Center in Rotterdam to work on the cytoskeleton and polarised cargo transport in neurons. In 2011, he became an assistant professor at Utrecht University, received a European Research Council starting grant and then a Vidi fellowship from the Netherlands Organisation for Scientific Research (NWO), and became associate professor in 2016. His group uses super-resolution microscopy and optogenetics to study how motor proteins achieve transport of cargo and organelles in different cell types.

Cx43 Isoform GJA1-20k Promotes Microtubule Dependent Mitochondrial Transport.

Connexin 43 (Cx43, encoded by GJA1) is a cell-cell communication gap junction protein expressed in all organ systems. It was recently found that GJA1 mRNA undergoes alternative translation to generate N-terminal truncated isoforms, of which GJA1-20k is the most abundant. Here we report a surprising finding that, unlike full length GJA1-43k, GJA1-20k has a strong tropism for mitochondria. Exploring function, we found that GJA1-20k appears to be an organelle chaperone and that overexpression of GJA1-20k is sufficient to rescue mitochondrial localization to the cell periphery upon exposure to hydrogen peroxide, which effectively limits the network fragmentation that occurs with oxidative stress. By high-resolution fluorescent imaging and electron microscopy, we determined that GJA1-20k is enriched at the interface between mitochondria and microtubules, appearing to load organelles for transport. Mutagenesis experiments revealed that although the microtubule-binding domain (MTBD) in GJA1-20k is not necessary for protein localization to mitochondria, the MTBD is essential for GJA1-20k to facilitate mitochondrial transport and maintain mitochondrial localization at the periphery. These results reveal an unexpected role for the alternatively translated isoform of the Cx43 gap junction protein, GJA1-20k, which is to facilitate microtubule-based mitochondrial transport and to maintain mitochondrial network integrity during cellular stress.

Cytoplasmic Flow and Mixing Due to Deformation of Motile Cells

The cytoplasm of a living cell is a dynamic environment through which intracellular components must move and mix. In motile, rapidly deforming cells such as human neutrophils, bulk cytoplasmic flow couples cell deformation to the transport and dispersion of cytoplasmic particles. Using particle-tracking measurements in live neutrophil-like cells, we demonstrate that fluid flow associated with the cell deformation contributes to the motion of small acidic organelles, dominating over diffusion on timescales above a few seconds. We then use a general physical model of particle dispersion in a deforming fluid domain to show that transport of organelle-sized particles between the cell periphery and the bulk can be enhanced by dynamic deformation comparable to that observed in neutrophils. Our results implicate an important mechanism contributing to organelle transport in these motile cells: cytoplasmic flow driven by cell shape deformation.

MTORC1/rpS6 regulates blood-testis barrier dynamics and spermatogenetic function in the testis in vivo.

The blood-testis barrier (BTB), conferred by Sertoli cells in the mammalian testis, is an important ultrastructure that supports spermatogenesis. Studies using animal models have shown that a disruption of the BTB leads to meiotic arrest, causing defects in spermatogenesis and male infertility. To better understand the regulation of BTB dynamics, we report findings herein to understand the role of ribosomal protein S6 (rpS6), a downstream signaling protein of mammalian target of rapamycin complex 1 (mTORC1), in promoting BTB disruption in the testis in vivo, making the barrier "leaky." Overexpression of wild-type rpS6 (rpS6-WT, the full-length cDNA cloned into the mammalian expression vector pCI-neo) and a constitutively active quadruple phosphomimetic mutant cloned into pCI-neo (p-rpS6-MT) vs. control (empty pCI-neo vector) was achieved by transfecting adult rat testes with the corresponding plasmid DNA using a Polyplus in vivo-jetPEI transfection reagent. On the basis of an in vivo functional BTB integrity assay, p-rpS6-MT was found to induce BTB disruption better than rpS6-WT did (and no effects in empty vector control), leading to defects in spermatogenesis, including loss of spermatid polarity and failure in the transport of cells (e.g., spermatids) and organelles (e.g., phagosomes), to be followed by germ exfoliation. More important, rpS6-WT and p-rpS6-MT exert their disruptive effects through changes in the organization of actin- and microtubule (MT)-based cytoskeletons, which are mediated by changes in the spatiotemporal expression of actin- and MT-based binding and regulatory proteins. In short, mTORC1/rpS6 signaling complex is a regulator of spermatogenesis and BTB by modulating the organization of the actin- and MT-based cytoskeletons.

Lipid remodelling in neuroendocrine secretion.

Neuroendocrine cells secrete hormones and polypeptides through a complex membrane trafficking process that involves the transport of specific organelles, called large dense core secretory granules, from the Golgi apparatus to specialised sites at the plasma membrane where these vesicles are successively exocytosed and recaptured by endocytosis through tightly coupled reactions. The minimal machinery required for exocytosis has been defined as SNARE proteins associated with few accessory proteins. On the other side, clathrin and dynamin constitute major components of some of the most important endocytotic pathways. Although many protein contributors of both exocytosis and endocytosis are now identified, their actual interplay is not well resolved. Furthermore, the necessary tight coupling of exocytosis and compensatory endocytosis to maintain membrane homeostasis in neuroendocrine cells is far from being understood. In this review, we focus on the more recently identified role of lipids in these important processes that are above all membrane remodelling events.

Wide phenotypic spectrum in axonal Charcot-Marie-Tooth neuropathy type 2 patients with KIF5A mutations.

The kinesin heavy chain isoform 5A (KIF5A) gene, which encodes a microtubule-based motor protein, plays an important role in the transport of organelles in the nerve cells. Mutations in the KIF5A showed a wide phenotypic spectrum from hereditary spastic paraplegia (HSP) to axonal Charcot-Marie-Tooth peripheral neuropathy type 2 (CMT2). This study identified three pathogenic KIF5A mutations in Korean CMT2 patients by whole exome sequencing. Two mutations (p.Arg204Trp and p.Arg280His) were previously reported, but p.Leu558Pro was determined to be a novel de novo mutation. All the mutations were not observed in the healthy controls and were located in highly conserved domains among vertebrate species. The p.Arg204Trp mutation was identified from a CMT2 patient with additional complex phenotypes of HSP, ataxia, fatigability and pyramidal sign, but the p.Arg280His and p.Leu588Pro mutations were identified in each axonal CMT2 patient. The p.Arg204Trp mutation was previously reported in a HSP patient with no CMT symptom. The p.Arg280His mutation was reported in a CMT2 patient, which was similarly with our case. However, it was also once reported in a HSP patient with pes cavus. As the first report in Korea, this study identified three KIF5A mutations as the underlying cause of axonal peripheral neuropathy with or without the HSP phenotype. We confirmed a wide inter- and intra-allelic phenotypic spectrum by the mutations in the KIF5A.

Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization

BackgroundDysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation.MethodsWe have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions.ResultsUsing highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function.ConclusionsThis report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by corroborating findings of others, and providing important new information on essential RPE cell biological properties.

Wide phenotypic spectrum of axonal peripheral neuropathy patients with KIF5A mutations

Background: The KIF5A gene encodes a microtubule-based motor protein (kinesin heavy chain isoform 5A) which plays an important role in the transport of organelles in the eukaryotic cells. Mutations in the KIF5A have been shown a wide phenotypic spectrum from hereditary spastic paraplegia (HSP) to axonal peripheral neuropathy.

Acetylated Microtubules Are Preferentially Bundled Leading to Enhanced Kinesin-1 Motility

The motor proteins kinesin and dynein transport organelles, mRNA, proteins, and signaling molecules along the microtubule cytoskeleton. In addition to serving as tracks for transport, the microtubule cytoskeleton directs intracellular trafficking by regulating the activity of motor proteins through the organization of the filament network, microtubule-associated proteins, and tubulin posttranslational modifications. However, it is not well understood how these factors influence motor motility, and in vitro assays and live cell observations often produce disparate results. To systematically examine the factors that contribute to cytoskeleton-based regulation of motor protein motility, we extracted intact microtubule networks from cells and tracked the motility of single fluorescently labeled motor proteins on these cytoskeletons. We find that tubulin acetylation alone does not directly affect kinesin-1 motility. However, acetylated microtubules are predominantly bundled, and bundling enhances kinesin run lengths and provides a greater number of available kinesin binding sites. The neuronal MAP tau is also not sensitive to tubulin acetylation, but enriches preferentially on highly curved regions of microtubules where it strongly inhibits kinesin motility. Taken together, these results suggest that the organization of the microtubule network is a key contributor to the regulation of motor-based transport.

Rab GTPases in Immunity and Inflammation.

Strict spatiotemporal control of trafficking events between organelles is critical for maintaining homeostasis and directing cellular responses. This regulation is particularly important in immune cells for mounting specialized immune defenses. By controlling the formation, transport and fusion of intracellular organelles, Rab GTPases serve as master regulators of membrane trafficking. In this review, we discuss the cellular and molecular mechanisms by which Rab GTPases regulate immunity and inflammation.

Single-Molecule Fluorescence Microscopy in Living Caenorhabditis elegans.

Transportation of organelles and biomolecules is vital for many cellular processes. Single-molecule (SM) fluorescence microscopy can expose molecular aspects of the dynamics that remain unresolved in ensemble experiments. For example, trajectories of individual, moving biomolecules can reveal velocity and changes therein, including pauses. We use SM imaging to study the dynamics of motor proteins and their cargo in the cilia of living C. elegans. To this end, we employ standard fluorescent proteins, an epi-illuminated, wide-field fluorescence microscope and mostly open-source software. This chapter describes the setup we use, the preparation of samples, a protocol for single-molecule imaging in C. elegans and data analysis.