Cytoskeletal Transport Research Articles

Motor protein traffic regulation by supply–demand balance of resources

In cells and in in vitro assays the number of motor proteins involved in biological transport processes is far from being unlimited. The cytoskeletal binding sites are in contact with the same finite reservoir of motors (either the cytosol or the flow chamber) and hence compete for recruiting the available motors, potentially depleting the reservoir and affecting cytoskeletal transport. In this work we provide a theoretical framework in which to study, analytically and numerically, how motor density profiles and crowding along cytoskeletal filaments depend on the competition of motors for their binding sites. We propose two models in which finite processive motor proteins actively advance along cytoskeletal filaments and are continuously exchanged with the motor pool. We first look at homogeneous reservoirs and then examine the effects of free motor diffusion in the surrounding medium. We consider as a reference situation recent in vitro experimental setups of kinesin-8 motors binding and moving along microtubule filaments in a flow chamber. We investigate how the crowding of linear motor proteins moving on a filament can be regulated by the balance between supply (concentration of motor proteins in the flow chamber) and demand (total number of polymerized tubulin heterodimers). We present analytical results for the density profiles of bound motors and the reservoir depletion, and propose novel phase diagrams that present the formation of jams of motor proteins on the filament as a function of two tuneable experimental parameters: the motor protein concentration and the concentration of tubulins polymerized into cytoskeletal filaments. Extensive numerical simulations corroborate the analytical results for parameters in the experimental range and also address the effects of diffusion of motor proteins in the reservoir. We then propose experiments for validating our models and discuss how the ‘supply–demand’ effects can regulate motor traffic also in in vivo conditions.

Malaria parasites and red cell variants

Multiple red cell variants are known to confer protection from malaria. Here, we review advances in identifying new variants that modulate malaria risk and in defining molecular mechanisms that mediate malaria protection. New red cell variants, including an innate variant in the red cell's major Ca²⁺ pump and the acquired state of iron deficiency, have been associated with protection from clinical falciparum malaria. The polymorphisms hemoglobin C (HbC) and hemoglobin S (HbS) - known to protect carriers from severe falciparum malaria - enhance parasite passage to mosquitoes and may promote malaria transmission. At the molecular level, substantial advances have been made in understanding the impact of HbS and HbC upon the interactions between host microRNAs and Plasmodium falciparum protein translation; remodeling of red cell cytoskeletal components and transport of parasite proteins to the red cell surface; and chronic activation of the human innate immune system, which induces tolerance to blood-stage parasites. Several polymorphisms have now been associated with protection from clinical vivax malaria or reduced Plasmodium vivax density, including Southeast Asian ovalocytosis and two common forms of glucose-6-phosphate dehydrogenase deficiency. Red cell variants that modulate malaria risk can serve as models to identify clinically relevant mechanisms of pathogenesis, and thus define parasite and host targets for next-generation therapies.

Progressive axonal transport and synaptic protein changes correlate with behavioral and neuropathological abnormalities in the heterozygous Q175 KI mouse model of Huntington's disease.

A long-term goal of modeling Huntington's disease (HD) is to recapitulate the cardinal features of the disease in mice that express both mutant and wild-type (WT) huntingtin (Htt), as HD commonly manifests as a heterozygous condition in humans, and loss of WT Htt is associated with loss-of-function. In a new heterozygous Q175 knock-in (KI) mouse model, we performed an extensive evaluation of motor and cognitive functional deficits, neuropathological and biochemical changes and levels of proteins involved in synaptic function, the cytoskeleton and axonal transport, at 1-16 months of age. Motor deficits were apparent at 6 months of age in Q175 KI mice and at that time, postmortem striatal gamma-aminobutyric acid (GABA) levels were elevated and mutant Htt inclusions were present throughout the brain. From 6 months of age, levels of proteins associated with synaptic function, including SNAP-25, Rab3A and PSD-95, and with axonal transport and microtubules, including KIF3A, dynein and dynactin, were altered in the striatum, motor cortex, prefrontal cortex and hippocampus of Q175 KI mice, compared with WT levels. At 12-16 months of age, Q175 KI mice displayed motor and cognitive deficits, which were paralleled at postmortem by striatal atrophy, cortical thinning, degeneration of medium spiny neurons, dense mutant Htt inclusion formation, decreased striatal dopamine levels and loss of striatal brain-derived neurotrophic factor (BDNF). Data from this study indicate that the heterozygous Q175 KI mouse represents a realistic model for HD and also provides new insights into the specific and progressive synaptic, cytoskeletal and axonal transport protein abnormalities that may accompany the disease.

Trafficking Highways to the Intercalated Disc: New Insights Unlocking the Specificity of Connexin 43 Localization

With each heartbeat, billions of cardiomyocytes work in concert to propagate the electrical excitation needed to effectively circulate blood. Regulated expression and timely delivery of connexin proteins to form gap junctions at the specialized cell-cell contact region, known as the intercalated disc, is essential to ventricular cardiomyocyte coupling. We focus this review on several regulatory mechanisms that have been recently found to govern the lifecycle of connexin 43 (Cx43), the short-lived and most abundantly expressed connexin in cardiac ventricular muscle. The Cx43 lifecycle begins with gene expression, followed by oligomerization into hexameric channels, and then cytoskeletal-based transport toward the disc region. Once delivered, hemichannels interact with resident disc proteins and are organized to effect intercellular coupling. We highlight recent studies exploring regulation of Cx43 localization to the intercalated disc, with emphasis on alternatively translated Cx43 isoforms and cytoskeletal transport machinery that together regulate Cx43 gap junction coupling between cardiomyocytes.

COMPARATIVE PROTEOMIC ANALYSIS OF EXHALED BREATH CONDENSATE IN PATIENTS WITH LUNG CARCINOMA USING HIGH RESOLUTION MASS-SPECTROMETRY

Analysis of exhaled breath condensate (EBC) is a promising non-invasive method to diagnose respiratory diseases. Most researchers emphasize the importance of proteomic analysis of EBC for early diagnosis of certain respiratory diseases including lung cancer. This study was aimed at identification of potential biomarkers of neoplastic disorders in EBC of patients with lung cancer using high-performance liquid chromatography and high resolution mass-spectrometry. The study involved 26 patients with lung carcinoma (21 males, 5 females, mean age 57 ± 12 years) and 23 healthy non-smokers (19 males, 4 females, mean age 30 ± 7 years). EBC samples were collected using a disposable portable condenser R-Tube. The most of proteins identified (65 %) belonged to keratin family including type 1 (1; 2; 5 and 6А) and type 2 (9; 10; 14; 16 and 17) cytoskeletal keratins and transport proteins (albumin, lipocalin-1). Keratin family proteins (5, 6 and 14) prevailed in lung cancer patients compared to controls ( p < 0.05). Other 6 proteins were also detected predominantly in lung cancer patients including b-subunit and a-subunit of haemoglobin, nuclear ubiquitous casein (NUCKS), high-mobility group proteins (HMG-I/HMG-Y), and lactoferrin. Most of these proteins could be used as a diagnostic panel to detect lung cancer. Further investigations are needed to estimate diagnostic values of these biomarkers and their role in pathogenesis of lung cancer.

Remote Control of Diverse Cytoskeletal Motors using Light-Activated Gearshifting

Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells. Protein engineering has been used to modify cytoskeletal motors for dynamic control of activity and directionality, providing direct tests of structure-function relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems. We have previously1 created myosin motors that can be signaled to switch directions in response to changes in [Ca2+]. Light is a more versatile control signal2 because it can be precisely modulated in space and time, and is generally orthogonal to cellular signaling. Here we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears - speed up, slow down, or switch directions - when exposed to blue light. Our structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. We have used in vitro motility assays to confirm robust spatiotemporal control over motor function and to characterize the kinetics of optical gearshifting. Our modular approach has yielded controllable motors for both actin-based and microtubule-based transport. Genetically encoded light-responsive motors will expand the optogenetics toolkit, complementing precise perturbations of ion channels and intracellular signaling with spatiotemporal control of cytoskeletal transport and contractility.1. Chen, L., Nakamura, M., Schindler, T.D., Parker, D. & Bryant, Z. Engineering controllable bidirectional molecular motors based on myosin. Nature nanotechnology 7, 252-6 (2012).2. Walter, W.J. & Diez, S. Myosin shifts into reverse gear. Nature nanotechnology7, 213-4 (2012).

Biomolecular motors in nanoscale materials, devices, and systems

Biomolecular motors are a unique class of intracellular proteins that are fundamental to a considerable number of physiological functions such as DNA replication, organelle trafficking, and cell division. The efficient transformation of chemical energy into useful work by these proteins provides strong motivation for their utilization as nanoscale actuators in ex vivo, meso- and macro-scale hybrid systems. Biomolecular motors involved in cytoskeletal transport are quite attractive models within this context due to their ability to direct the transport of nano-/micro-scale objects at rates significantly greater than diffusion, and in the absence of bulk fluid flow. As in living organisms, biomolecular motors involved in cytoskeletal transport (i.e., kinesin, dynein, and myosin) function outside of their native environment to dissipatively self-assemble biological, biomimetic, and hybrid nanostructures that exhibit nonequilibrium behaviors such as self-healing. These systems also provide nanofluidic transport function in hybrid nanodevices where target analytes are actively captured, sorted, and transported for autonomous sensing and analytical applications. Moving forward, the implementation of biomolecular motors will continue to enable a wide range of unique functionalities that are presently limited to living systems, and support the development of nanoscale systems for addressing critical engineering challenges.

ERK2-Mediated Phosphorylation of Par3 Regulates Neuronal Polarization

Axon formation is one of the most important events in neuronal polarization and is regulated by signaling molecules involved in cytoskeletal rearrangement and protein transport. We previously found that Partition-defective 3 (Par3) is associated with KIF3A (kinesin-2) and is transported into the nascent axon in a KIF3A-dependent fashion. Par3 interacts with the Rac-specific guanine nucleotide-exchange factors (GEFs) Tiam1/2, which activate Rac1, and participates in axon formation in cultured hippocampal neurons. However, the regulatory mechanism of the Par3-KIF3A interaction is poorly understood, and the role of Par3 in neuronal polarization in vivo remains elusive. Here, we found that extracellular signal-regulated kinase 2 (ERK2) directly interacts with Par3, that ERK2 phosphorylates Par3 at Ser-1116, and that the phosphorylated Par3 accumulates at the axonal tips in a manner dependent upon ERK2 activity. The phosphorylation of Par3 by ERK2 inhibited the interaction of Par3 with KIF3A but not with the other Par3 partners, including Par6 and aPKC. The phosphomimic mutant of Par3 (Par3-S1116D) showed less binding activity with the KIF3s and slower transport in the axons. The knockdown of Par3 by RNA interference impaired neuronal polarization, which was rescued with RNAi-resistant Par3, but not with the phosphomimic Par3 mutant, in cultured rat hippocampal neurons and mouse cortical projection neurons in vivo. These results suggest that ERK2 phosphorylates Par3 and inhibits its binding with KIF3A, thereby controlling Par3 transport and neuronal polarity.

Exclusion processes on networks as models for cytoskeletal transport

We present a study of exclusion processes on networks as models for complex transport phenomena, and in particular for active transport of motor proteins along the cytoskeleton. Specifically, we focus on the totally asymmetric simple exclusion process (TASEP) as well as its generalizations including backstepping (partially asymmetric simple exclusion process (PASEP)) and exchange with a bulk concentration (TASEP with Langmuir kinetics (TASEP-LK)). We build on the previously used effective rate approach to establish a general methodology in terms of effective rate diagrams, which allows for a simple classification of the stationary transport state of the total network. This approach is general and reveals generic features of exclusion processes on networks. Based on the three examples considered here, we show that the classification can be made in terms of three qualitative different network regimes: a homogeneous regime, a heterogeneous network regime and a heterogeneous segment regime. Using parameters representative of real motor proteins, we show how the transitions between these regimes can be regulated through a variety of multi-scale factors, such as the interplay of exclusion interactions, the non-equilibrium nature of the transport process, motor processivity and the network topology. Using the equilibrium limits of PASEP and TASEP-LK, we also shed further light on the emergence of density heterogeneities in active transport phenomena.

Anomalous Diffusion of Single Particles in Cytoplasm

The crowded intracellular environment poses a formidable challenge to experimental and theoretical analyses of intracellular transport mechanisms. Our measurements of single-particle trajectories in cytoplasm and their random-walk interpretations elucidate two of these mechanisms: molecular diffusion in crowded environments and cytoskeletal transport along microtubules. We employed acousto-optic deflector microscopy to map out the three-dimensional trajectories of microspheres migrating in the cytosolic fraction of a cellular extract. Classical Brownian motion (BM), continuous time random walk, and fractional BM were alternatively used to represent these trajectories. The comparison of the experimental and numerical data demonstrates that cytoskeletal transport along microtubules and diffusion in the cytosolic fraction exhibit anomalous (nonFickian) behavior and posses statistically distinct signatures. Among the three random-walk models used, continuous time random walk provides the best representation of diffusion, whereas microtubular transport is accurately modeled with fractional BM.

Mutations in CLC-5 cause disturbances in cytoskeletal dynamics and solute transport in Dent's disease renal proximal tubule cell-lines

Mutations in CLC-5 cause disturbances in cytoskeletal dynamics and solute transport in Dent's disease renal proximal tubule cell-lines

Calpastatin Overexpression Protects Axonal Transport in anIn VivoModel of Traumatic Axonal Injury

Traumatic brain injury (TBI) causes substantial morbidity and mortality worldwide. A key component of both mild and severe TBI is diffuse axonal injury. Except in cases of extreme mechanical strain, when axons are torn at the moment of trauma, axonal stretch injury is characterized by early cytoskeletal proteolysis, transport disruption, and secondary axotomy. Calpains, a family of Ca(2+)-dependent proteases, have been implicated in this pathologic cascade, but direct in vivo evidence is lacking. To test the hypothesis that calpains play a causal role in axonal stretch injury in vivo, we used our rat optic nerve stretch model following adeno-associated viral (AAV) vector-mediated overexpression of the endogenous calpain inhibitor calpastatin in optic nerve axons. AAV vectors were designed for optimal expression of human calpastatin (hCAST) in retinal ganglion cells (RGCs). Calpain inhibition by the expressed protein was then confirmed in primary cortical cultures. Finally, we performed bilateral intravitreal injections of AAV vectors expressing hCAST or the reporter protein ZsGreen 3 weeks prior to unilateral optic nerve stretch. Immediately after stretch injury, Fluoro-Gold was injected into the superior colliculi for assessment of retrograde axonal transport. Rats were euthanized 4 days after stretch injury. Both hCAST and ZsGreen were detected in axons throughout the optic nerve to the chiasm. Calpastatin overexpression partially preserved axonal transport after stretch injury (58.3±15.6% reduction in Fluoro-Gold labeling relative to uninjured contralateral controls in ZsGreen-expressing RGCs, versus 33.8±23.9% in hCAST-expressing RGCs; p=0.038). These results provide direct evidence that axonal calpains play a causal role in transport disruption after in vivo stretch injury.

Comprehensive proteomic analysis of human dentin

Proteomic analysis of the human body is a significant recent scientific endeavour. In this study, we investigated the proteomic profile of human dentin using modern analytical and mass spectrometric techniques. Five healthy permanent human molars from five adults were cut, pulverized, denaturated with guanidine buffer, and demineralized with EDTA buffer. The extracted proteins were analysed by gel electrophoresis (SDS-PAGE and two-dimensional gel electrophoresis), digested with trypsin, and separated by liquid chromatography/high-resolution tandem mass spectrometry. We identified 289 proteins with high confidence, 90 of which had not been previously detected in human dentin. Nine (currently hypothetical) proteins were identified for the first time in an actual human sample. The proteins have a variety of functions, including calcium-ion binding, formation of the extracellular matrix, formation of the cytoskeleton, cytoskeletal protein binding, immune response, and transport. In conclusion, this is the first use of two-dimensional electrophoresis for investigating human dentin.

Loss of Cytoskeletal Transport during Egress Critically Attenuates Ectromelia Virus Infection In Vivo

Egress of wrapped virus (WV) to the cell periphery following vaccinia virus (VACV) replication is dependent on interactions with the microtubule motor complex kinesin-1 and is mediated by the viral envelope protein A36. Here we report that ectromelia virus (ECTV), a related orthopoxvirus and the causative agent of mousepox, encodes an A36 homologue (ECTV-Mos-142) that is highly conserved despite a large truncation at the C terminus. Deleting the ECTV A36R gene leads to a reduction in the number of extracellular viruses formed and to a reduced plaque size, consistent with a role in microtubule transport. We also observed a complete loss of virus-associated actin comets, another phenotype dependent on A36 expression during VACV infection. ECTV ΔA36R was severely attenuated when used to infect the normally susceptible BALB/c mouse strain. ECTV ΔA36R replication and spread from the draining lymph nodes to the liver and spleen were significantly reduced in BALB/c mice and in Rag-1-deficient mice, which lack T and B lymphocytes. The dramatic reduction in ECTV ΔA36R titers early during the course of infection was not associated with an augmented immune response. Taken together, these findings demonstrate the critical role that subcellular transport pathways play not only in orthopoxvirus infection in an in vitro context but also during orthopoxvirus pathogenesis in a natural host. Furthermore, despite the attenuation of the mutant virus, we found that infection nonetheless induced protective immunity in mice, suggesting that orthopoxvirus vectors with A36 deletions may be considered another safe vaccine alternative.

Global Effects of Adriamycin Treatment on Mouse Splenic Protein Levels

Adriamycin (ADR) is a potent anticancer drug used to treat a variety of cancers. Patients treated with ADR have experienced side effects such as heart failure, cardiomyopathy, and "chemobrain", which have been correlated to changes in protein expression in the heart and brain. In order to better understand cellular responses that are disrupted following ADR treatment in immune tissues, this work focuses on spleen. Significantly reduced spleen sizes were found in ADR-treated mice. Global isotopic labeling of tryptic peptides and nanoflow reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) were employed to determine differences in the relative abundances of proteins from ADR-treated mice relative to controls. Fifty-nine proteins of the 388 unique proteins identified showed statistically significant differences in expression levels following acute ADR treatment. Differentially expressed proteins are involved in processes such as cytoskeletal structural integrity, cellular signaling and transport, transcription and translation, immune response, and Ca(2+) binding. These are the first studies to provide insight to the downstream effects of ADR treatment in a peripheral immune organ such as spleen using proteomics.

Colchicine for the Prevention of Postoperative Atrial Fibrillation

Colchicine for the Prevention of Postoperative Atrial Fibrillation

Messengers, motors and mysteries: sorting of eukaryotic mRNAs by cytoskeletal transport

It has become increasingly apparent in recent years that the subcellular localization of specific mRNAs is a prevalent method for spatially controlling gene expression. In most cases, targeting of mRNAs is mediated by transport along cytoskeletal filaments by molecular motors. However, the means by which specific messages are recognized and linked to the motors are poorly understood. Here, I will provide an overview of recent progress in elucidating the molecular mechanisms and principles of mRNA transport, including several studies highlighting the co-operation of different motors during the localization process. Important outstanding questions will also be highlighted.

Alpha‐ and beta‐synucleins are new diagnostic tools for acute erythroid leukemia and acute megakaryoblastic leukemia

α-Synuclein is a key component of the Lewy body, a large globular protein complex that forms in the nervous system of patients with Parkinson disease and other dementias [1-3]. Since α-synuclein also occurs in megakaryocytic and erythroid lineages [4-7], we wondered what role synucleins had in the hematopoietic system. Therefore, we studied the expression of α-, β-, and γ-synucleins in a comprehensive panel of patient bone marrows and leukemic cell lines. We observed under expression of α-synuclein in the megakaryocytes of myeloproliferative neoplasm (MPN), but not normal reactive marrow (NRM) or myelodysplastic syndrome (MDS). Conversely, we observed over expression of β-synuclein in the blasts of megakaryoblastic leukemias (MegL), but not acute myeloid leukemia (AML) or erythroleukemia (EryL), suggesting that α- and β-synucleins could be useful adjunct markers for the early detection of MDS and the differential diagnosis of EryL and MegL from other AMLs.

Differential Gene Expression in Daphnia magna Suggests Distinct Modes of Action and Bioavailability for ZnO Nanoparticles and Zn Ions

Zinc oxide nanoparticles (ZnO NPs) are being rapidly developed for use in consumer products, wastewater treatment, and chemotherapy providing several possible routes for ZnO NP exposure to humans and aquatic organisms. Recent studies have shown that ZnO NPs undergo rapid dissolution to Zn(2+), but the relative contribution of Zn(2+) to ZnO NP bioavailability and toxicity is not clear. We show that a fraction of the ZnO NPs in suspension dissolves, and this fraction cannot account for the toxicity of the ZnO NP suspensions to Daphnia magna. Gene expression profiling of D. magna exposed to ZnO NPs or ZnSO(4) at sublethal concentrations revealed distinct modes of toxicity. There was also little overlap in gene expression between ZnO NPs and SiO(x) NPs, suggesting specificity for the ZnO NP expression profile. ZnO NPs effected expression of genes involved in cytoskeletal transport, cellular respiration, and reproduction. A specific pattern of differential expression of three biomarker genes including a multicystatin, ferritin, and C1q containing gene were confirmed for ZnO NP exposure and provide a suite of biomarkers for identifying environmental exposure to ZnO NPs and differentiating between NP and ionic exposure.

Actin cables and the exocyst form two independent morphogenesis pathways in the fission yeast

Cell morphogenesis depends on polarized exocytosis. One widely held model posits that long-range transport and exocyst-dependent tethering of exocytic vesicles at the plasma membrane sequentially drive this process. Here, we describe that disruption of either actin-based long-range transport and microtubules or the exocyst did not abolish polarized growth in rod-shaped fission yeast cells. However, disruption of both actin cables and exocyst led to isotropic growth. Exocytic vesicles localized to cell tips in single mutants but were dispersed in double mutants. In contrast, a marker for active Cdc42, a major polarity landmark, localized to discreet cortical sites even in double mutants. Localization and photobleaching studies show that the exocyst subunits Sec6 and Sec8 localize to cell tips largely independently of the actin cytoskeleton, but in a cdc42 and phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂)-dependent manner. Thus in fission yeast long-range cytoskeletal transport and PIP₂-dependent exocyst represent parallel morphogenetic modules downstream of Cdc42, raising the possibility of similar mechanisms in other cell types.