Vladimir Rodionov

Functional coordination of microtubule-based and actin-based motility in melanophores

The fish melanophore has been considered the exemplar of microtubule-based organelle transport. In this system, a radial array of uniformly polarized microtubules [1] provides a framework on which dynein-related and kinesin-related motors drive pigment granules toward the minus or plus ends, respectively [2-4]. Stimulation of minus-end motors accounts satisfactorily for aggregation of granules at the cell center. Rapid dispersion is clearly microtubule-dependent; however, the uniform distribution of granules throughout the cytoplasm is paradoxical because stimulation of plus-end motors is predicted to drive the granules to the cell margin. This paradox suggested that the transport system was incompletely understood. Here, we report the discovery of a microtubule-independent motility system in fish melanophores. The system is based on actin filaments and is required for achieving uniform distribution of pigment granules. When it is abrogated, granules accumulate at the cells margin as predicted for microtubule plus-end motors acting alone. The results presented here demonstrate the functional coordination of microtubule and actin filament systems, a finding that may be of general significance for organelle motility in cytoplasm.

Centrosome positioning in interphase cells

The position of the centrosome is actively maintained at the cell center, but the mechanisms of the centering force remain largely unknown. It is known that centrosome positioning requires a radial array of cytoplasmic microtubules (MTs) that can exert pushing or pulling forces involving MT dynamics and the activity of cortical MT motors. It has also been suggested that actomyosin can play a direct or indirect role in this process. To examine the centering mechanisms, we introduced an imbalance of forces acting on the centrosome by local application of an inhibitor of MT assembly (nocodazole), and studied the resulting centrosome displacement. Using this approach in combination with microinjection of function-blocking probes, we found that a MT-dependent dynein pulling force plays a key role in the positioning of the centrosome at the cell center, and that other forces applied to the centrosomal MTs, including actomyosin contractility, can contribute to this process.

Switching between Microtubule- and Actin-Based Transport Systems in Melanophores Is Controlled by cAMP Levels

BACKGROUND Intracellular transport involves the movement of organelles along microtubules (MTs) or actin filaments (AFs) by means of opposite-polarity MT motors or actin-dependent motors of the myosin family. The correct delivery of organelles to their different destinations involves a precise coordination of the two transport systems. Such coordination could occur through regulation of the densities of the two cytoskeletal systems or through regulation of the activities of the cytoskeletal motors by signaling mechanisms. RESULTS To investigate the mechanisms of switching between MT and AF-dependent transport, we examine the influence of the densities of the MT and AF network on pigment transport in fish melanophores. We also change signaling by using activators and inhibitors of Protein Kinase A (PKA). We find that the key parameters characterizing pigment granule transport along MTs do not depend on MT density and are not significantly altered by complete disruption of AFs. In contrast, the kinetics of changes in these parameters correlate with the kinetics of changes in the intracellular levels of cAMP and are affected by the inhibitors of PKA, suggesting the regulation of MT- and AF-dependent motors by cAMP-induced signaling. Furthermore, perturbation of cAMP levels prevents the transfer of pigment granules from MTs onto AFs. CONCLUSIONS We conclude that the switching of pigment granules between the two major cytoskeletal systems is independent of the densities of MT or AF but is tightly controlled by signaling events.

Investigating Sub-Spine Actin Dynamics in Rat Hippocampal Neurons with Super-Resolution Optical Imaging

Morphological changes in dendritic spines represent an important mechanism for synaptic plasticity which is postulated to underlie the vital cognitive phenomena of learning and memory. These morphological changes are driven by the dynamic actin cytoskeleton that is present in dendritic spines. The study of actin dynamics in these spines traditionally has been hindered by the small size of the spine. In this study, we utilize a photo-activation localization microscopy (PALM)–based single-molecule tracking technique to analyze F-actin movements with ∼30-nm resolution in cultured hippocampal neurons. We were able to observe the kinematic (physical motion of actin filaments, i.e., retrograde flow) and kinetic (F-actin turn-over) dynamics of F-actin at the single-filament level in dendritic spines. We found that F-actin in dendritic spines exhibits highly heterogeneous kinematic dynamics at the individual filament level, with simultaneous actin flows in both retrograde and anterograde directions. At the ensemble level, movements of filaments integrate into a net retrograde flow of ∼138 nm/min. These results suggest a weakly polarized F-actin network that consists of mostly short filaments in dendritic spines.

Self-organization of a radial microtubule array by dynein-dependent nucleation of microtubules.

Polarized radial arrays of cytoplasmic microtubules (MTs) with minus ends clustered at the cell center define the organization of the cytoplasm through interaction with microtubule motors bound to membrane organelles or chromosomes. It is generally assumed that the radial organization results from nucleation of MTs at the centrosome. However, radial MT array can also be attained through self-organization that requires the activity of a minus-end-directed MT motor, cytoplasmic dynein. In this study we examine the role of cytoplasmic dynein in the self-organization of a radial MT array in cytoplasmic fragments of fish melanophores lacking the centrosome. After activation of dynein motors bound to membrane-bound organelles, pigment granules, the fragments rapidly form polarized radial arrays of MTs and position pigment aggregates at their centers. We show that rearrangement of MTs in the cytoplasm is achieved through dynein-dependent MT nucleation. The radial pattern is generated by continuous disassembly and reassembly of MTs and concurrent minus-end-directed transport of pigment granules bearing the nucleation sites.

CLIP-170-Dependent Capture of Membrane Organelles by Microtubules Initiates Minus-End Directed Transport

Cytoplasmic microtubules (MTs) continuously grow and shorten at free plus ends. During mitosis, this dynamic behavior allows MTs to capture chromosomes to initiate their movement to the spindle poles; however, the role of MT dynamics in capturing organelles for transport in interphase cells has not been demonstrated. Here we use Xenopus melanophores to test the hypothesis that MT dynamics significantly contribute to the efficiency of MT minus-end directed transport of membrane organelles. We demonstrate that initiation of transport of membrane-bounded melanosomes (pigment granules) to the cell center involves their capture by MT plus ends, and that inhibition of MT dynamics or loss of the MT plus-end tracking protein CLIP-170 from MT tips dramatically inhibits pigment aggregation. We conclude that MT dynamics are required for the initiation of MT transport of membrane organelles in interphase cells, and that +TIPs such as CLIP-170 play an important role in this process.

Loss of Spastin Function Results in Disease-Specific Axonal Defects in Human Pluripotent Stem Cell-Based Models of Hereditary Spastic Paraplegia

Human neuronal models of hereditary spastic paraplegias (HSP) that recapitulate disease‐specific axonal pathology hold the key to understanding why certain axons degenerate in patients and to developing therapies. SPG4, the most common form of HSP, is caused by autosomal dominant mutations in the SPAST gene, which encodes the microtubule‐severing ATPase spastin. Here, we have generated a human neuronal model of SPG4 by establishing induced pluripotent stem cells (iPSCs) from an SPG4 patient and differentiating these cells into telencephalic glutamatergic neurons. The SPG4 neurons displayed a significant increase in axonal swellings, which stained strongly for mitochondria and tau, indicating the accumulation of axonal transport cargoes. In addition, mitochondrial transport was decreased in SPG4 neurons, revealing that these patient iPSC‐derived neurons recapitulate disease‐specific axonal phenotypes. Interestingly, spastin protein levels were significantly decreased in SPG4 neurons, supporting a haploinsufficiency mechanism. Furthermore, cortical neurons derived from spastin‐knockdown human embryonic stem cells (hESCs) exhibited similar axonal swellings, confirming that the axonal defects can be caused by loss of spastin function. These spastin‐knockdown hESCs serve as an additional model for studying HSP. Finally, levels of stabilized acetylated‐tubulin were significantly increased in SPG4 neurons. Vinblastine, a microtubule‐destabilizing drug, rescued this axonal swelling phenotype in neurons derived from both SPG4 iPSCs and spastin‐knockdown hESCs. Thus, this study demonstrates the successful establishment of human pluripotent stem cell‐based neuronal models of SPG4, which will be valuable for dissecting the pathogenic cellular mechanisms and screening compounds to rescue the axonal degeneration in HSP. Stem Cells 2014;32:414–423

Finding the Cell Center by a Balance of Dynein and Myosin Pulling and Microtubule Pushing: A Computational Study

By comparing computer modeling predictions with observations, we conclude that strong dynein and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center.

Centering of a radial microtubule array by translocation along microtubules spontaneously nucleated in the cytoplasm

Positioning of a radial array of microtubules (MTs) in the cell centre is crucial for cytoplasmic organization, but the mechanisms of such centering are difficult to study in intact cells that have pre-formed radial arrays. Here, we use cytoplasmic fragments of melanophores, and cytoplasts of BS-C-1 cells to study MT centering mechanisms. Using live imaging and computer modelling, we show that the MT aster finds a central location in the cytoplasm by moving along spontaneously nucleated non-astral MTs towards a point at which MT nucleation events occur equally on all sides. We hypothesize that similar mechanisms, in the presence of the centrosome, contribute to this centering mechanism and ensure the robustness of cytoplasmic organization.

Force-dependent detachment of kinesin-2 biases track switching at cytoskeletal filament intersections.

Intracellular trafficking of organelles often involves cytoskeletal track switching. Organelles such as melanosomes are transported by multiple motors including kinesin-2, dynein, and myosin-V, which drive switching between microtubules and actin filaments during dispersion and aggregation. Here, we used optical trapping to determine the unitary and ensemble forces of kinesin-2, and to reconstitute cargo switching at cytoskeletal intersections in a minimal system with kinesin-2 and myosin-V motors bound to beads. Single kinesin-2 motors exerted forces up to ∼5 pN, similar to kinesin-1. However, kinesin-2 motors were more likely to detach at submaximal forces, and the duration of force maintenance was short as compared to kinesin-1. In multimotor assays, force increased with kinesin-2 density but was not affected by the presence of myosin-V. In crossed filament assays, switching frequencies of motor-bound beads were dependent on the starting track. At equal average forces, beads tended to switch from microtubules onto overlying actin filaments consistent with the relatively faster detachment of kinesin-2 at near-maximal forces. Thus, in addition to relative force, switching probability at filament intersections is determined by the dynamics of motor-filament interaction, such as the quick detachment of kinesin-2 under load. This may enable fine-tuning of filament switching in the cell.