An integral protein of the inner nuclear membrane localizes to the mitotic spindle in mammalian cells

Abstract

Here, we characterize a transmembrane protein of the nuclear envelope that we name spindle-associated membrane protein 1 (Samp1). The protein is conserved in metazoa and fission yeast and is homologous to Net5 in rat and Ima1 in Schizosaccharomyces pombe. We show that, in human cells, the protein is a membrane-spanning polypeptide with an apparent molecular mass of 43 kDa. This is consistent with a predicted polypeptide of 392 amino acids that has five transmembrane segments and its C-terminus exposed to the nucleoplasm. During interphase, Samp1 was specifically distributed in the inner nuclear membrane. Post-transcriptional silencing of Samp1 expression resulted in separation of centrosomes from the nuclear envelope, indicating that it is functionally connected to the cytoskeleton. At the onset of mitosis, most of the protein dispersed out into the ER, as expected. However, during mitosis, a significant fraction of the protein specifically localized to the polar regions of the mitotic spindle. We demonstrate for the first time, in human cells, the existence of a membranous structure overlapping with the mitotic spindle. Interestingly, another integral inner nuclear membrane protein, emerin, was absent from the spindle-associated membranes. Thus, Samp1 defines a specific membrane domain associated with the mitotic spindle.

Samp1 is functionally associated with the LINC complex and A-type lamina networks

“…We have previously shown that Samp1 is an integral membrane protein located in the inner nuclear membrane of many cultured cells (Buch et al, 2009). Careful inspection by confocal fluorescence microscopy of equatorial, as well as oblique, optical sections of HeLa cell nuclei stained using antibodies specific for Samp1 revealed that Samp1 was not uniformly distributed in the INM.…”

“…Anchoring of centrosomes close to the nucleus was also shown to depend on the novel transmembrane INM protein Samp1 (transmembrane protein 201) (Buch et al, 2009), suggesting that Samp1 is also connected to LINC complexes. As highlighted in a recent review (Wilson and Berk, 2010), anchorage of the microtubule cytoskeleton at the nuclear surface is particularly important for the relative positioning of centrosomes versus nuclei during cell migration (Gomes et al, 2005) or during interkinetic nuclear migration when neuronal cells divide (Burke and Roux, 2009;Starr, 2009) in the hippocampus and cerebellum of the developing mammalian brain.…”

Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope

“…Instead, we pre-cross-linked cellular components with dithiobis (succinimidyl propionate), prepared the nuclei pellet (free of NE; SI Appendix, Fig. S2) (34,35), and solubilized proteins from pellet as the starting material for all IP experiments in this report.…”

TAN lines

“…60 The inner nuclear membrane protein, Ima1, interacts with centromeric heterochromatin and is thought to help distribute the forces generated by microtubules through the LINC complex throughout the nuclear envelope allowing for productive force transduction. It will be interesting to see if the mammalian homologue of Ima1 (NET-5/Samp1), 61 or other heterochromatin interacting proteins are important for TAN line formation and function.…”