Jen Hsuan Wei

ERK regulates Golgi and centrosome orientation towards the leading edge through GRASP65

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.

Nucleoporin Levels Regulate Cell Cycle Progression and Phase-Specific Gene Expression

The Nup107-160 complex, the largest subunit of the nuclear pore, is multifunctional. It mediates mRNA export in interphase, and has roles in kinetochore function, spindle assembly, and postmitotic nuclear pore assembly. We report here that the levels of constituents of the Nup107-160 complex are coordinately cell cycle-regulated. At mitosis, however, a member of the complex, Nup96, is preferentially downregulated. This occurs via the ubiquitin-proteasome pathway. When the levels of Nup96 are kept high, a significant delay in G1/S progression occurs. Conversely, in cells of Nup96(+/-) mice, which express low levels of Nup96, cell cycle progression is accelerated. These lowered levels of Nup96 yield specific defects in nuclear export of certain mRNAs and protein expression, among which are key cell cycle regulators. Thus, Nup96 levels regulate differential gene expression in a phase-specific manner, setting the stage for proper cell cycle progression.

Mitotic division of the mammalian Golgi apparatus.

Successful cell reproduction requires faithful duplication and proper segregation of cellular contents, including not only the genome but also intracellular organelles. Since the Golgi apparatus is an essential organelle of the secretory pathway, its accurate inheritance is therefore of importance to sustain cellular function. Regulation of Golgi division and its coordination with cell cycle progression involves a series of sequential events that are subjected to a precise spatiotemporal control. Here, we summarize the current knowledge about the underlying mechanisms, the molecular players and the biological relevance of this process, particularly in mammalian cells, and discuss the unsolved problems and future perspectives opened by the recent studies.

Unraveling the Golgi ribbon.

The Golgi apparatus lies at the heart of the secretory pathway where it receives, modifies and sorts protein cargo to the proper intracellular or extracellular location. Although this secretory function is highly conserved throughout the eukaryotic kingdom, the structure of the Golgi complex is arranged very differently among species. In particular, Golgi membranes in vertebrate cells are integrated into a single compact entity termed the Golgi ribbon that is normally localized in the perinuclear area and in close vicinity to the centrosomes. This organization poses a challenge for cell division when the single Golgi ribbon needs to be partitioned into the two daughter cells. To ensure faithful inheritance in the progeny, the Golgi ribbon is divided in three consecutive steps in mitosis, namely disassembly, partitioning and reassembly. However, the structure of the Golgi ribbon is only present in higher animals and Golgi disassembly during mitosis is not ubiquitous in all organisms. Therefore, there must be unique reasons to build up the Golgi in this particular conformation and to preserve it over generations. In this review, we first highlight the diversity of the Golgi architecture in different organisms and revisit the concept of the Golgi ribbon. Following on, we discuss why the ribbon is needed and how it forms in vertebrate cells. Lastly, we conclude with likely purposes of mitotic ribbon disassembly and further propose mechanisms by which it regulates mitosis.

Golgi Cisternal Unstacking Stimulates COPI Vesicle Budding and Protein Transport

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.

The mitotic spindle mediates inheritance of the Golgi ribbon structure

The mammalian Golgi ribbon disassembles during mitosis and reforms in both daughter cells after division. Mitotic Golgi membranes concentrate around the spindle poles, suggesting that the spindle may control Golgi partitioning. To test this, cells were induced to divide asymmetrically with the entire spindle segregated into only one daughter cell. A ribbon reforms in the nucleated karyoplasts, whereas the Golgi stacks in the cytoplasts are scattered. However, the scattered Golgi stacks are polarized and transport cargo. Microinjection of Golgi extract together with tubulin or incorporation of spindle materials rescues Golgi ribbon formation. Therefore, the factors required for postmitotic Golgi ribbon assembly are transferred by the spindle, but the constituents of functional stacks are partitioned independently, suggesting that Golgi inheritance is regulated by two distinct mechanisms.

Modular organization of the mammalian Golgi apparatus

The Golgi apparatus is essential for post-translational modifications and sorting of proteins in the secretory pathway. In addition, it further performs a broad range of specialized functions. This functional diversity is achieved by combining basic morphological modules of cisternae into higher ordered structures. Linking cisternae into stacks that are further connected through tubules into a continuous Golgi ribbon greatly increases its efficiency and expands its repertoire of functions. During cell division, the different modules of the Golgi are inherited by different mechanisms to maintain its functional and morphological composition.

Spatial separation of Golgi and ER during mitosis protects SREBP from unregulated activation

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.

GM130 Regulates Golgi-Derived Spindle Assembly by Activating TPX2 and Capturing Microtubules

Spindle assembly requires the coordinated action of multiple cellular structures to nucleate and organize microtubules in a precise spatiotemporal manner. Among them, the contributions of centrosomes, chromosomes, and microtubules have been well studied, yet the involvement of membrane-bound organelles remains largely elusive. Here, we provide mechanistic evidence for a membrane-based, Golgi-derived microtubule assembly pathway in mitosis. Upon mitotic entry, the Golgi matrix protein GM130 interacts with importin α via a classical nuclear localization signal that recruits importin α to the Golgi membranes. Sequestration of importin α by GM130 liberates the spindle assembly factor TPX2, which activates Aurora-A kinase and stimulates local microtubule nucleation. Upon filament assembly, nascent microtubules are further captured by GM130, thus linking Golgi membranes to the spindle. Our results reveal an active role for the Golgi in regulating spindle formation to ensure faithful organelle inheritance.

Vesicular stomatitis virus inhibits mitotic progression and triggers cell death

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.