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.
DOCK7 (dedicator of cytokinesis 7) is a guanidine nucleotide exchange factor (GEF) for Rac1 GTPase that is involved in neuronal polarity and axon generation as well in Schwann cell differentiation and myelination. Recently, we identified DOCK7 as the binding partner of unconventional myosin VI (MVI) in neuronal-lineage PC12 cells and postulated that this interaction could be important in vivo [Majewski et al. (2012) Biochem Cell Biol., 90:565-574]. Herein, we found that MVI-DOCK7 interaction takes also place in other cell lines and demonstrated that MVI cargo domain via its RRL motif binds to DOCK7 C-terminal M2 and DHR2 domains. In MVI knockdown cells, lower Rac1 activity and a decrease of DOCK7 phosphorylation on Tyr1118 were observed, indicating that MVI could contribute to DOCK7 activity. MVI and DOCK7 co-localization was maintained during NGF-stimulated PC12 cell differentiation and observed also in the outgrowths. Also, during differentiation an increase in phosphorylation of DOCK7 as well as of its downstream effector JNK kinase was detected. Interestingly, overexpression of GFP-tagged MVI cargo domain (GFP-GT) impaired protrusion formation indicating that full length protein is important for this process. Moreover, a transient increase in Rac activity observed at 5min of NGF-stimulated differentiation of PC12 cells (overexpressing either GFP or GFP-MVI) was not detected in cells overexpressing the cargo domain. These data indicate that MVI-DOCK7 interaction could have functional implications in the protrusion outgrowth, and full length MVI seems to be important for delivery and maintenance of DOCK7 along the protrusions, and exerting its GEF activity.
Capillary zone electrophoretic (CZE) analysis of monomeric prolamins (wheat gliadins and rye secalins) covered 28 hexaploid triticale ( Triticosecale x Wittm.) cultivars. The ethanol-soluble proteins were separated on an uncoated fused-silica capillary using the isoelectric 60 mM iminodiacetic (IDA) buffer in conjunction with 20% (v/v) acetonitrile and 0.075% (w/v) polyvinylpyrrolidone (PVP). For each separation, dynamic coating of the capillary wall with a buffer containing 0.1 M IDA and 0.05% (w/v) hydroxypropylmethylcellulose (HPMC) was performed. Separations of prolamins provided very good resolution and high reproducibility (<0.8% RSD). Prolamin profiles of all analyzed cultivars showed both qualitative and quantitative differences, including number of peaks, presence or absence of peaks, and area of peaks. The number of prolamin peaks detected in particular triticale cultivars varied from 22 to 28; in total, 56 components were distinguished. The CZE electropherograms of prolamins showed five main groups of protein peaks, in order of mobility alpha-prolamins, beta-prolamins, gamma-prolamins, omega1-prolamins, and omega2-prolamins, with migration times of 6.8-7.7, 7.8-10.4, 10.5-12.2, 12.3-17.4, and 17.5-25.6 min, respectively. Triticale seeds in comparison with wheat contained fewer alpha-prolamins and higher quantity of omega-prolamins. Hierarchical clustering of the investigated cultivars was based on Bhattacharyya distances calculated from the CZE data. The cultivars grouped in four main clusters. The obtained CZE results were compared with A-PAGE data.
Miozyny to oddziałujące z aktyną białka motoryczne, zaangażowane w skurcz mięśni, migrację komórek i transport wewnątrzkomórkowy. Występują one we wszystkich organizmach eukariotycznych, w tym w pierwotniakach i roślinach. Miozyny zbudowane są z jednego lub dwóch łańcuchów ciężkich oraz kilku łańcuchów lekkich (1-7 na łańcuch ciężki). Zidentyfikowano kilka tysięcy sekwencji łańcuchów ciężkich miozyn występujących w kilkuset gatunkach. W łańcuchach ciężkich miozyn wyróżniono główkę obejmującą domenę motoryczną (miejsce oddziaływania z aktyną i wiązania ATP) i szyjkę z motywami IQ (miejsce niekowalencyjnego wiązania się z łańcuchami lekkimi) oraz ogonek (zawierający domeny warunkujące specyficzne funkcje poszczególnych izoform miozyny). Na podstawie różnic w sekwencji aminokwasowej domeny motorycznej wyróżniono ponad trzydzieści rodzin w nadrodzinie miozyn, z czego 12, reprezentowanych przez 40 izoform, występuje u człowieka. Miozyny mięśniowe (tworzące rodzinę II) zwane są konwencjonalnymi, a pozostałe - niekonwencjonalnymi. Niniejszy artykuł opisuje nadrodzinę miozyn, a w szczególności budowę i funkcje tych miozyn niekonwencjonalnych, które są obecne w komórkach miogennych i mięśniach poprzecznie-prążkowanych.
Aktyna i miozyna to białka kojarzone przede wszystkim z ich kluczową rolą w generacji skurczu mięśni. Natomiast poza izoformami charakterystycznymi dla mięśni są również izoformy aktyny i miozyny, które występują we wszystkich typach komórek i tkanek (patrz artykuł Suszek i współaut. w tym zeszycie KOSMOSU). Badania prowadzone w ostatnich dwóch dekadach wykazały niezbicie, że zarówno aktyna (i szereg białek wiążących aktynę) oraz liczne miozyny (przedstawiciele rodzin I, II, V, VI, XVI i XVIII) lokalizują się w jądrze komórkowym gdzie są zaangażowane w procesy transkrypcji i naprawy DNA, transport w nukleoplazmie oraz import i eksport jądrowy, a także w utrzymywanie architektury jądra. Niniejszy artykuł opisuje dotychczasowy stan wiedzy o roli układu akto-miozynowego w jądrze komórkowym.
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