The Golgi apparatus in mammalian cells is composed of flattened cisternae that are densely packed to form stacks. We have used the Golgi stacking protein GRASP65 as a tool to modify the stacking state of Golgi cisternae. We established an assay to measure protein transport to the cell surface in post-mitotic cells in which the Golgi was unstacked. Cells with an unstacked Golgi showed a higher transport rate compared to cells with stacked Golgi membranes. Vesicle budding from unstacked cisternae in vitro was significantly increased compared to stacked membranes. These results suggest that Golgi cisternal stacking can directly regulate vesicle formation and thus the rate of protein transport through the Golgi. The results further suggest that at the onset of mitosis, unstacking of cisternae allows extensive and rapid vesiculation of the Golgi in preparation for its subsequent partitioning.

Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that reside as inactive precursors in the endoplasmic reticulum (ER) membrane. After sterol depletion, the proteins are transported to the Golgi apparatus, where they are cleaved by site-1 protease (S1P). Cleavage releases the active transcription factors, which then enter the nucleus to induce genes that regulate cellular levels of cholesterol and phospholipids. This regulation depends on the spatial separation of the Golgi and the ER, as mixing of the compartments induces unregulated activation of SREBPs. Here, we show that S1P is localized to the Golgi, but cycles continuously through the ER and becomes trapped when ER exit is inhibited. During mitosis, S1P is associated with mitotic Golgi clusters, which remain distinct from the ER. In mitotic cells, S1P is active, but SREBP is not cleaved as S1P and SREBP reside in different compartments. Together, these results indicate that the spatial separation of the Golgi and the ER is maintained during mitosis, which is essential to protect the S1P substrate SREBP from unregulated activation during mitosis.

Enhanced telomere maintenance is evident in malignant cancers. While telomeres are thought to be inherently heterochromatic, detailed mechanisms of how epigenetic modifications impact telomere protection and structures are largely unknown in human cancers. Here we develop a molecular tethering approach to experimentally enrich heterochromatin protein HP1α specifically at telomeres. This results in increased deposition of H3K9me3 at cancer cell telomeres. Telomere extension by telomerase is attenuated, and damage-induced foci at telomeres are reduced, indicating augmentation of telomere stability. Super-resolution STORM imaging shows an unexpected increase in irregularity of telomeric structure. Telomere-tethered chromo shadow domain (CSD) mutant I165A of HP1α abrogates both the inhibition of telomere extension and the irregularity of telomeric structure, suggesting the involvement of at least one HP1α-ligand in mediating these effects. This work presents an approach to specifically manipulate the epigenetic status locally at telomeres to uncover insights into molecular mechanisms underlying telomere structural dynamics.

Directed cell migration requires the orientation of the Golgi and centrosome toward the leading edge. We show that stimulation of interphase cells with the mitogens epidermal growth factor or lysophosphatidic acid activates the extracellular signal-regulated kinase (ERK), which phosphorylates the Golgi structural protein GRASP65 at serine 277. Expression of a GRASP65 Ser277 to alanine mutant or a GRASP65 1-201 truncation mutant, neither of which can be phosphorylated by ERK, prevents Golgi orientation to the leading edge in a wound assay. We show that phosphorylation of GRASP65 with recombinant ERK leads to the loss of GRASP65 oligomerization and causes Golgi cisternal unstacking. Furthermore, preventing Golgi polarization by expressing mutated GRASP65 inhibits centrosome orientation, which is rescued upon disassembly of the Golgi structure by brefeldin A. We conclude that Golgi remodeling, mediated by phosphorylation of GRASP65 by ERK, is critical for the establishment of cell polarity in migrating cells.

Vesicular stomatitis virus (VSV) infects and kills a wide range of cell types; however, the mechanisms involved in VSV-mediated cell death are not fully understood. Here we show that VSV infection interferes with mitotic progression, resulting in cell death. This effect requires the interaction of VSV matrix (M) protein with the Rae1-Nup98 complex in mitosis, which is associated with a subset of ribonucleoproteins (RNPs). VSV displaced Rae1 from spindle poles, caused spindle abnormalities and triggered substantial cell death during metaphase. These effects were attenuated in cells infected with VSV expressing a mutant M protein that does not bind efficiently to the Rae1-Nup98-RNP complex. In cells that progressed to late mitosis, M protein prevented proper nuclear formation and chromatin decondensation. VSV is an oncolytic (anti-tumour) agent as it preferentially replicates and kills tumour cells. As tumour cells have a high mitotic index, VSV-mediated mitotic cell death probably contributes to its oncolytic activity.