Rachel Santarella

HURP Is Part of a Ran-Dependent Complex Involved in Spindle Formation

BACKGROUND GTP-loaded Ran induces the assembly of microtubules into aster-like and spindle-like structures in Xenopus egg extract. The microtubule-associated protein (MAP), TPX2, can mediate Rans role in aster formation, but factors responsible for the transition from aster-like to spindle-like structures have not been described. RESULTS Here we identify a complex that is required for the conversion of aster-like to spindle-like structures. The complex consists of two characterized MAPs (TPX2, XMAP215), a plus end-directed motor (Eg5), a mitotic kinase (Aurora A), and HURP, a protein associated with hepatocellular carcinoma. Formation and function of the complex is dependent on Aurora A activity. HURP protein was further characterized and shown to bind microtubules and affect their organization both in vitro and in vivo. In egg extract, anti-HURP antibodies disrupt the formation of both Ran-dependent and chromatin and centrosome-induced spindles. HURP is also required for the proper formation and function of mitotic spindles in HeLa cells. CONCLUSIONS HURP is a new and essential component of the mitotic apparatus. HURP acts as part of a multicomponent complex that affects the growth or stability of spindle MTs and is required for spindle MT organization.

MEL-28/ELYS is required for the recruitment of nucleoporins to chromatin and postmitotic nuclear pore complex assembly

The metazoan nuclear envelope (NE) breaks down and re‐forms during each cell cycle. Nuclear pore complexes (NPCs), which allow nucleocytoplasmic transport during interphase, assemble into the re‐forming NE at the end of mitosis. Using in vitro NE assembly, we show that the vertebrate homologue of MEL‐28 (maternal effect lethal), a recently discovered NE component in Caenorhabditis elegans, functions in postmitotic NPC assembly. MEL‐28 interacts with the Nup107–160 complex (Nup for nucleoporin), an important building block of the NPC, and is essential for the recruitment of the Nup107–160 complex to chromatin. We suggest that MEL‐28 acts as a seeding point for NPC assembly.

Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly

Barrier‐to‐autointegration factor (BAF) is an essential, highly conserved, metazoan protein. BAF interacts with LEM (LAP2, emerin, MAN1) domain‐carrying proteins of the inner nuclear membrane. We analyzed the in vivo function of BAF in Caenorhabditis elegans embryos using both RNA interference and a temperature‐sensitive baf‐1 gene mutation and found that BAF is directly involved in nuclear envelope (NE) formation. NE defects were observed independent of and before the chromatin organization phenotype previously reported in BAF‐depleted worms and flies. We identified vaccinia‐related kinase (VRK) as a regulator of BAF phosphorylation and localization. VRK localizes both to the NE and chromatin in a cell‐cycle‐dependent manner. Depletion of VRK results in several mitotic defects, including impaired NE formation and BAF delocalization. We propose that phosphorylation of BAF by VRK plays an essential regulatory role in the association of BAF with chromatin and nuclear membrane proteins during NE formation.

A role for gp210 in mitotic nuclear-envelope breakdown

The cytoplasmic and nuclear compartments of animal cells mix during mitosis on disassembly of the nuclear envelope (NE). NE breakdown (NEBD) involves the dispersion of the nuclear membranes and associated proteins, including nuclear pore complexes (NPCs) and the nuclear lamina. Among the approximately 30 NPC components known, few contain transmembrane domains. gp210 is a single-pass transmembrane glycoprotein of metazoan NPCs. We show that both RNAi-mediated depletion and mutation of Caenorhabditis elegans gp210 affect NEBD in early embryonic cells, preventing lamin depolymerization and leading to the formation of twinned nuclei after mitosis owing to physical interference with normal chromosome alignment and segregation. When added to in vitro assembled nuclei, antibodies specific for the C-terminal cytoplasmic tail of gp210 completely blocked NEBD. This treatment inhibited mitotic hyper-phosphorylation of gp210. Phosphorylation of gp210 is proposed to be mediated by cyclin-B–cdc2 and we show that depletion of cyclin B from C. elegans embryos also leads to a nuclear-twinning phenotype. In summary, we show that gp210 is important for efficient NPC disassembly and NEBD and suggest that phosphorylation of gp210 is an early event in NEBD that is required for lamin disassembly and other aspects of NEBD.

Nup53 Is Required for Nuclear Envelope and Nuclear Pore Complex Assembly

Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53-Nup155 complex plays a critical role in the processes of NPC and NE assembly.

Physical and chemical perturbations induce the formation of protein aggregates with different structural features

The in vitro aggregation of the model GST-GFP fusion protein was induced by several effectors, including those mimicking variations occurring under cell stress conditions. In particular, we examined the effects of thermal treatments, redox state and pH variations, salt addition, and freezing and thawing cycles. The resulting aggregates displayed different morphologies as seen by electron microscopy, and different secondary and tertiary structures, as indicated by Fourier transform infrared spectroscopy and fluorescence. Therefore, proteins can be forced to undergo multiple aggregation pathways that lead to assemblies with different molecular structures and, possibly, specific physiological and pathological roles. In conclusion, great caution should be taken in inferring conclusions on protein aggregation and disaggregation in vivo from results obtained using aggregates produced under non-physiological perturbations.

Electron microscopy of lamin and the nuclear lamina in Caenorhabditis elegans.

The nuclear lamina is found between the inner nuclear membrane and the peripheral chromatin. Lamins are the main components of the nuclear lamina, where they form protein complexes with integral proteins of the inner nuclear membrane, transcriptional regulators, histones and chromatin modifiers. Lamins are required for mechanical stability, chromatin organization, Pol II transcription, DNA replication, nuclear assembly, and nuclear positioning. Mutations in human lamins cause at least 13 distinct human diseases, collectively termed laminopathies, affecting muscle, adipose, bone, nerve and skin cells, and range from muscular dystrophies to accelerated aging. Caenorhabditis elegans has unique advantages in studying lamins and nuclear lamina genes including low complexity of lamina genes and the unique ability of bacterially expressed C. elegans lamin protein to form stable 10 nm fibers. In addition, transgenic techniques, simple application of RNA interference, sophisticated genetic analyses, and the production of a large collection of mutant lines, all make C. elegans especially attractive for studying the functions of its nuclear lamina genes. In this chapter we will include a short review of our current knowledge of nuclear lamina in C. elegans and will describe electron microscopy techniques used for their analyses.