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

Abstract

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.

Dynein and muskelin control myosin VI delivery towards the neuronal nucleus

“…The presented data connect to previous findings of elevated nuclear myosin VI levels after stimulation of mammalian cell lines (Hari-Gupta et al, 2020;Jung et al, 2006;Majewski et al, 2018). They extend these observations by showing that at least a prominent portion of myosin VI depends on an active transfer toward the mammalian nucleus.…”

Myosin VI moves on nuclear actin filaments and supports long-range chromatin rearrangements

“…This long bound fraction of MVI might correspond to a potential tethering function of nuclear MVI, analogous to its tethering functions in the cytoplasm (Avraham et al, 1995;Warner et al, 2003). Without further study, we cannot assign the binding time classes to particular processes, binding partners or structures, although possible candidates could be the already established partners of nuclear MVI (Majewski et al, 2018), such as RNA polymerase II (Fili et al, 2017;Vreugde et al, 2006), NDP52 (Fili et al, 2017) and DNA (Fili et al, 2017). Chromatin exhibits significant organizational order.…”

Formation of Aberrant Myotubes by Myoblasts Lacking Myosin VI Is Associated with Alterations in the Cytoskeleton Organization, Myoblast Adhesion and Fusion

“…It is noteworthy that this is in agreement with our earlier report on the C2C12 cell line with MVI knockdown [ 13 ]. The mechanisms of the observed changes in the expression levels of the myogenic factors remain to be unveiled, but one possibility is that MVI could participate in transcription, as was shown for several cancer cell lines, including neurosecretory rat pheochromocytoma PC12 cells [ 24 , 28 ]. However, this is rather unlikely as, in contrast to numerous cancer cells, MVI is not present in myoblast nuclei [ 13 ].…”

“…Snell’s waltzer mice ( SV ) lacking functional MVI also have several other defects, such as cardiac hypertrophy, as well as brain, kidney and spermiogenesis dysfunctions [ 11 , 12 , 19 , 20 , 21 , 22 ]. MVI is involved in endocytosis and the intracellular transport of vesicles and organelles, cell migration, the maintenance of Golgi apparatus, actin cytoskeleton organization, and possibly in gene transcription [ 23 , 24 , 25 , 26 , 27 , 28 ]. MVI fulfills its functions through interaction with actin (binding to the N-terminal motor domain in the ATP-dependent manner), and its partner proteins (binding to the C-terminal cargo domain through a positively charged RRL region and a hydrophobic WWY region) [ 29 ].…”