Increasing evidence suggests functional compartmentalization of interphase nuclei. This includes preferential interior localization of gene-rich and early replicating chromosome regions versus peripheral localization of gene-poor and late replicating chromosome regions , association of some active genes with nuclear speckles or transcription "factories", and association of transcriptionally repressed genes with heterochromatic regions. Dynamic changes in chromosome compartmentalization imply mechanisms for long-range interphase chromatin movements. However, live cell imaging in mammalian cells has revealed limited chromatin mobility, described as "constrained diffusion". None of these studies, though, have examined a chromosome locus undergoing an inducible repositioning between two different nuclear compartments. Here we demonstrate migration of an interphase chromosome site from the nuclear periphery to the interior 1-2 hr after targeting a transcriptional activator to this site. Spot redistribution is perturbed by specific actin or nuclear myosin I mutants. Extended periods of chromosome immobility are interspersed with several minute periods in which chromosomes move unidirectionally along curvilinear paths oriented roughly perpendicular to the nuclear envelope at velocities of 0.1-0.9 microm/min over distances of 1-5 microm. Our results suggest an active mechanism for fast and directed long-range interphase chromosome movements dependent directly or indirectly on actin/myosin.

Long-Range Directional Movement of an Interphase Chromosome Site Dependent on Actin and Nuclear Myosin

Myosin VI-Dependent Actin Cages Encapsulate Parkin-Positive Damaged Mitochondria

Mechanical Regulation of Transcription: Recent Advances.

The intracellular functions of myosin motors requires a number of adaptor molecules, which control cargo attachment, but also fine‐tune motor activity in time and space. These motor–adaptor–cargo interactions are often weak, transient or highly regulated. To overcome these problems, we use a proximity labelling‐based proteomics strategy to map the interactome of the unique minus end‐directed actin motor MYO6. Detailed biochemical and functional analysis identified several distinct MYO6‐adaptor modules including two complexes containing RhoGEFs: the LIFT (LARG‐Induced F‐actin for Tethering) complex that controls endosome positioning and motility through RHO‐driven actin polymerisation; and the DISP (DOCK7‐Induced Septin disPlacement) complex, a novel regulator of the septin cytoskeleton. These complexes emphasise the role of MYO6 in coordinating endosome dynamics and cytoskeletal architecture. This study provides the first in vivo interactome of a myosin motor protein and highlights the power of this approach in uncovering dynamic and functionally diverse myosin motor complexes.

The MYO6 interactome reveals adaptor complexes coordinating early endosome and cytoskeletal dynamics.

Objective- Human and mouse megakaryocytes lacking NBEAL2 (neurobeachin-like 2) produce platelets where α-granules lack protein cargo. This cargo is mostly megakaryocyte-synthesized, but some proteins, including FGN (fibrinogen), are endocytosed. In this study, we examined the trafficking of both types of cargo within primary megakaryocytes cultured from normal and NBEAL2-null mice, to determine the role of NBEAL2 in α-granule maturation. We also examined the interaction of NBEAL2 with the granule-associated protein P-selectin in human megakaryocytes and platelets. Approach and Results- Fluorescence microscopy was used to compare uptake of labeled FGN by normal and NBEAL2-null mouse megakaryocytes, which was similar in both. NBEAL2-null cells, however, showed decreased FGN retention, and studies with biotinylated protein showed rapid loss rather than increased degradation. Intracellular tracking via fluorescence microscopy revealed that in normal megakaryocytes, endocytosed FGN sequentially associated with compartments expressing RAB5 (Ras-related protein in brain 5), RAB7 (Ras-related protein in brain 7), and P-selectin, where it was retained. A similar initial pattern was observed in NBEAL2-null megakaryocytes, but then FGN passed from the P-selectin compartment to RAB11 (Ras-related protein in brain 11)-associated endosomes before release. Megakaryocyte-synthesized VWF (Von Willebrand factor) was observed to follow the same route out of NBEAL2-null cells. Immunofluorescence microscopy revealed intracellular colocalization of NBEAL2 with P-selectin in human megakaryocytes, proplatelets, and platelets. Native NBEAL2 and P-selectin were coimmunoprecipitated from platelets and megakaryocytes. Conclusions- NBEAL2 is not required for FGN uptake by megakaryocytes. NBEAL2 is required for the retention of both endocytosed and megakaryocyte-synthesized proteins by maturing α-granules, and possibly by platelet-borne granules. This function may involve interaction of NBEAL2 with P-selectin.

https://www.ahajournals.org/doi/pdf/10.1161/ATVBAHA.118.311270

NBEAL2 (Neurobeachin-Like 2) Is Required for Retention of Cargo Proteins by α-Granules During Their Production by Megakaryocytes.

Interaction of myosin VI and its binding partner DOCK7 plays an important role in NGF-stimulated protrusion formation in PC12 cells.

Myosin VI (MVI) is a unique actin-based motor protein moving towards the minus end of actin filaments, in the opposite direction than other known myosins. Besides well described functions of MVI in endocytosis and maintenance of Golgi apparatus, there are few reports showing its involvement in transcription. We previously demonstrated that in neurosecretory PC12 cells MVI was present in the cytoplasm and nucleus, and its depletion caused substantial inhibition of cell migration and proliferation. Here, we show an increase in nuclear localization of MVI upon cell stimulation, and identification of potential nuclear localization (NLS) and nuclear export (NES) signals within MVI heavy chain. These signals seem to be functional as the MVI nuclear presence was affected by the inhibitors of nuclear import (ivermectin) and export (leptomycin B). In nuclei of stimulated cells, MVI colocalized with active RNA polymerase II, BrUTP-containing transcription sites and transcription factor SP1 as well as SC35 and PML proteins, markers of nuclear speckles and PML bodies, respectively. Mass spectrometry analysis of samples of a GST-pull-down assay with the MVI tail domain as a "bait" identified several new potential MVI binding partners. Among them are proteins involved in transcription and post-transcriptional processes. We confirmed interaction of MVI with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and nucleolin, proteins involved in pre-mRNA binding and transport, and nucleolar function, respectively. Our data provide an insight into mechanisms of involvement of MVI in nuclear processes via interaction with nuclear proteins and support a notion for important role(s) for MVI in gene expression.

https://www.tandfonline.com/doi/pdf/10.1080/19491034.2017.1421881

Myosin VI in the nucleus of neurosecretory PC12 cells: Stimulation-dependent nuclear translocation and interaction with nuclear proteins*

Actin and myosins are the proteins mainly known from their key roles in muscle contraction. However, besides typical muscle isoforms there are actins and myosins that are present in all cell and tissue types. Studies performed within the last two decades have irrefutably shown that both the cytoplasmic actin isoforms (along with numerous actin-binding proteins) as well as many myosins (representing class I, II, V, VI, XVI and XVIII) are present within the nucleus. They play important roles in nuclear processes as they are involved in transcription and DNA repair, intranuclear transport as well as nuclear import and export, and in maintenance of nuclear architecture. This article describes the current knowledge on the acto-myosin system in this biggest cellular compartment.

/pdf/aktyna-i-miozyny-w-jadrze-komorkowym-15s6is4tkr.pdf

Aktyna i miozyny w jądrze komórkowym

Myosins, actin-dependent molecular motors, are engaged in muscle contraction, cell migration and intracellular transport. They are present in all eukaryotic organisms including protists and plants. They are composed of one or two heavy chains, and a number of light chains (1-7 per a heavy chain). Several thousands of myosin heavy chains have been sequenced in hundreds of species. The heavy chain is composed of a motor domain (with actin and ATP binding sites), a neck with IQ motifs (where light chains bind to) and a tail (with domains determining specific functions of a given myosin). A myosin superfamily is divided into over 30 families based on differences in the motor domain primary sequence. Twelve families represented by 40 isoforms are expressed in humans. Well known muscle myosins forming a family II are termed as conventional while all others are termed as unconventional. The article describes the myosin superfamily with emphasis on structure and function of unconventional myosins present  in myogenic cells and striated muscles.

/pdf/miozyny-niekonwencjonalne-i-ich-rola-w-miesniach-poprzecznie-1zsgig829y.pdf

Jolanta Nowak

Papers

Interaction of myosin VI and its binding partner DOCK7 plays an important role in NGF-stimulated protrusion formation in PC12 cells.

Myosin VI in the nucleus of neurosecretory PC12 cells: Stimulation-dependent nuclear translocation and interaction with nuclear proteins*

Aktyna i miozyny w jądrze komórkowym

Miozyny niekonwencjonalne i ich rola w mięśniach poprzecznie prążkowanych i komórkach miogennych