A protein domain-based interactome network for C. elegans early embryogenesis.

Many protein-protein interactions are mediated through independently folding modular domains. Proteome-wide efforts to model protein-protein interaction or "interactome" networks have largely ignored this modular organization of proteins. We developed an experimental strategy to efficiently identify interaction domains and generated a domain-based interactome network for proteins involved in C. elegans early-embryonic cell divisions. Minimal interacting regions were identified for over 200 proteins, providing important information on their domain organization. Furthermore, our approach increased the sensitivity of the two-hybrid system, resulting in a more complete interactome network. This interactome modeling strategy revealed insights into C. elegans centrosome function and is applicable to other biological processes in this and other organisms.

Microtubules as key coordinators of nuclear envelope and endoplasmic reticulum dynamics during mitosis.

During mitosis, cells comprehensively restructure their interior to promote the faithful inheritance of DNA and cytoplasmic contents. In metazoans, this restructuring entails disassembly of the nuclear envelope, redistribution of its components into the endoplasmic reticulum (ER) and eventually nuclear envelope reassembly around the segregated chromosomes. The microtubule cytoskeleton has recently emerged as a critical regulator of mitotic nuclear envelope and ER dynamics. Microtubules and associated molecular motors tear open the nuclear envelope in prophase and remove nuclear envelope remnants from chromatin. Additionally, two distinct mechanisms of microtubule-based regulation of ER dynamics operate later in mitosis. First, association of the ER with microtubules is reduced, preventing invasion of ER into the spindle area, and second, organelle membrane is actively cleared from metaphase chromosomes. However, we are only beginning to understand the role of microtubules in shaping and distributing ER and other organelles during mitosis.

Reticulon-like REEP4 at the inner nuclear membrane promotes nuclear pore complex formation.

Nuclear pore complexes (NPCs) are channels within the nuclear envelope that mediate nucleocytoplasmic transport. NPCs form within the closed nuclear envelope during interphase or assemble concomitantly with nuclear envelope reformation in late stages of mitosis. Both interphase and mitotic NPC biogenesis require coordination of protein complex assembly and membrane deformation. During early stages of mitotic NPC assembly, a seed for new NPCs is established on chromatin, yet the factors connecting the NPC seed to the membrane of the forming nuclear envelope are unknown. Here, we report that the reticulon homology domain protein REEP4 not only localizes to high-curvature membrane of the cytoplasmic endoplasmic reticulum but is also recruited to the inner nuclear membrane by the NPC biogenesis factor ELYS. This ELYS-recruited pool of REEP4 promotes NPC assembly and appears to be particularly important for NPC formation during mitosis. These findings suggest a role for REEP4 in coordinating nuclear envelope reformation with mitotic NPC biogenesis.

REEP3 and REEP4 determine the tubular morphology of the endoplasmic reticulum during mitosis.

The endoplasmic reticulum (ER) is extensively remodeled during metazoan open mitosis. However, whether the ER becomes more tubular or more cisternal during mitosis is controversial, and dedicated factors governing the morphology of the mitotic ER have remained elusive. Here, we describe the ER membrane proteins REEP3 and REEP4 as major determinants of ER morphology in metaphase cells. REEP3/4 are specifically required for generating the high-curvature morphology of mitotic ER and promote ER tubulation through their reticulon homology domains (RHDs). This ER-shaping activity of REEP3/4 is distinct from their previously described function to clear ER from metaphase chromatin. We further show that related REEP proteins do not contribute to mitotic ER shaping and provide evidence that the REEP3/4 carboxyterminus mediates regulation of the proteins. These findings confirm that ER converts to higher curvature during mitosis, identify REEP3/4 as specific and crucial morphogenic factors mediating ER tubulation during mitosis, and define the first cell cycle-specific role for RHD proteins.

A versatile multivariate image analysis pipeline reveals features of Xenopus extract spindles.

Imaging datasets are rich in quantitative information. However, few cell biologists possess the tools necessary to analyze them. Here, we present a large dataset of Xenopusextract spindle images together with an analysis pipeline designed to assess spindle morphology across a range of experimental conditions. Our analysis of different spindle types illustrates how kinetochore microtubules amplify spindle microtubule density. Extract mixing experiments reveal that some spindle features titrate, while others undergo switch-like transitions, and multivariate analysis shows the pleiotropic morphological effects of modulating the levels of TPX2, a key spindle assembly factor. We also apply our pipeline to analyze nuclear morphology in human cell culture, showing the general utility of the segmentation approach. Our analyses provide new insight into the diversity of spindle types and suggest areas for future study. The approaches outlined can be applied by other researchers studying spindle morphology and adapted with minimal modification to other experimental systems.

REEP3/4 ensure endoplasmic reticulum clearance from metaphase chromatin and proper nuclear envelope architecture.

Dynamic interactions between membrane-bound organelles and the microtubule cytoskeleton are crucial to establish, maintain, and remodel the internal organization of cells throughout the cell cycle. However, the molecular nature of these interactions remains poorly understood. We performed a biochemical screen for microtubule-membrane linkers and identified REEP4, a previously uncharacterized endoplasmic reticulum (ER) protein. Depletion of REEP4 and the closely related REEP3 from HeLa cells causes defects in cell division and a proliferation of intranuclear membranes derived from the nuclear envelope. This phenotype originates in mitosis, when ER membranes accumulate on metaphase chromosomes. Microtubule binding and mitotic ER clearance from chromosomes both depend on a short, positively charged amino acid sequence connecting the two hydrophobic domains of REEP4. Our results show that REEP3/4 function redundantly to clear the ER from metaphase chromatin, thereby ensuring correct progression through mitosis and proper nuclear envelope architecture.

Nucleocytoplasmic shuttling of the Golgi phosphatidylinositol 4-kinase Pik1 is regulated by 14-3-3 proteins and coordinates Golgi function with cell growth.

The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p-14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling.

The C. elegans RSA complex localizes protein phosphatase 2A to centrosomes and regulates mitotic spindle assembly.

Microtubule behavior changes during the cell cycle and during spindle assembly. However, it remains unclear how these changes are regulated and coordinated. We describe a complex that targets the Protein Phosphatase 2A holoenzyme (PP2A) to centrosomes in C. elegans embryos. This complex includes Regulator of Spindle Assembly 1 (RSA-1), a targeting subunit for PP2A, and RSA-2, a protein that binds and recruits RSA-1 to centrosomes. In contrast to the multiple functions of the PP2A catalytic subunit, RSA-1 and RSA-2 are specifically required for microtubule outgrowth from centrosomes and for spindle assembly. The centrosomally localized RSA-PP2A complex mediates these functions in part by regulating two critical mitotic effectors: the microtubule destabilizer KLP-7 and the C. elegans regulator of spindle assembly TPXL-1. By regulating a subset of PP2A functions at the centrosome, the RSA complex could therefore provide a means of coordinating microtubule outgrowth from centrosomes and kinetochore microtubule stability during mitotic spindle assembly.