A genome-scale resource for in vivo tag-based protein function exploration in C. elegans.

Understanding the in vivo dynamics of protein localization and their physical interactions is important for many problems in biology. To enable systematic protein function interrogation in a multicellular context, we built a genome-scale transgenic platform for in vivo expression of fluorescent- and affinity-tagged proteins in Caenorhabditis elegans under endogenous cis regulatory control. The platform combines computer-assisted transgene design, massively parallel DNA engineering, and next-generation sequencing to generate a resource of 14,637 genomic DNA transgenes, which covers 73% of the proteome. The multipurpose tag used allows any protein of interest to be localized in vivo or affinity purified using standard tag-based assays. We illustrate the utility of the resource by systematic chromatin immunopurification and automated 4D imaging, which produced detailed DNA binding and cell/tissue distribution maps for key transcription factor proteins.

Centrosomes. Regulated assembly of a supramolecular centrosome scaffold in vitro.

The centrosome organizes microtubule arrays within animal cells and comprises two centrioles surrounded by an amorphous protein mass called the pericentriolar material (PCM). Despite the importance of centrosomes as microtubule-organizing centers, the mechanism and regulation of PCM assembly are not well understood. In Caenorhabditis elegans, PCM assembly requires the coiled-coil protein SPD-5. We found that recombinant SPD-5 could polymerize to form micrometer-sized porous networks in vitro. Network assembly was accelerated by two conserved regulators that control PCM assembly in vivo, Polo-like kinase-1 and SPD-2/Cep192. Only the assembled SPD-5 networks, and not unassembled SPD-5 protein, functioned as a scaffold for other PCM proteins. Thus, PCM size and binding capacity emerge from the regulated polymerization of one coiled-coil protein to form a porous network.

The Caenorhabditis elegans pericentriolar material components SPD-2 and SPD-5 are monomeric in the cytoplasm before incorporation into the PCM matrix.

Centrosomes are the main microtubule-organizing centers in animal cells. Centrosomes consist of a pair of centrioles surrounded by a matrix of pericentriolar material (PCM) that assembles from cytoplasmic components. In Caenorhabditis elegans embryos, interactions between the coiled-coil proteins SPD-5 and SPD-2 and the kinase PLK-1 are critical for PCM assembly. However, it is not known whether these interactions promote the formation of cytoplasmic complexes that are added to the PCM or whether the components interact only during incorporation into the PCM matrix. Here we address this problem by using a combination of live-cell fluorescence correlation spectroscopy, mass spectrometry, and hydrodynamic techniques to investigate the native state of PCM components in the cytoplasm. We show that SPD-2 is monomeric, and neither SPD-2 nor SPD-5 exists in complex with PLK-1. SPD-5 exists mostly as a monomer but also forms complexes with the PP2A-regulatory proteins RSA-1 and RSA-2, which are required for microtubule organization at centrosomes. These results suggest that the interactions between SPD-2, SPD-5, and PLK-1 do not result in formation of cytoplasmic complexes, but instead occur in the context of PCM assembly.

Limiting amounts of centrosome material set centrosome size in C. elegans embryos.

BACKGROUND: The ways in which cells set the size of intracellular structures is an important but largely unsolved problem [1]. Early embryonic divisions pose special problems in this regard. Many checkpoints common in somatic cells are missing from these divisions, which are characterized by rapid reductions in cell size and short cell cycles [2]. Embryonic cells must therefore possess simple and robust mechanisms that allow the size of many of their intracellular structures to rapidly scale with cell size. RESULTS: Here, we study the mechanism by which one structure, the centrosome, scales in size during the early embryonic divisions of C. elegans. We show that centrosome size is directly related to cell size and is independent of lineage. Two findings suggest that the total amount of maternally supplied centrosome proteins could limit centrosome size. First, the combined volume of all centrosomes formed at any one time in the developing embryo is constant. Second, the total volume of centrosomes in any one cell is independent of centrosome number. By increasing the amount of centrosome proteins in the cell, we provide evidence that one component that limits centrosome size is the conserved pericentriolar material protein SPD-2 [3], which we show binds to and targets polo-like kinase 1 [3, 4] to centrosomes. CONCLUSIONS: We propose a limiting component hypothesis, in which the volume of the cell sets centrosome size by limiting the total amount of centrosome components. This idea could be a general mechanism for setting the size of intracellular organelles during development.

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.

The mbk-2 kinase is required for inactivation of MEI-1/katanin in the one-cell Caenorhabditis elegans embryo.

The Caenorhabditis elegans early embryo is widely used to study the regulation of microtubule-related processes. In a screen for mutants affecting the first cell division, we isolated a temperature-sensitive mutation affecting pronuclear migration and spindle positioning, phenotypes typically linked to microtubule or centrosome defects. In the mutant, microtubules are shorter and chromosome segregation is impaired, while centrosome organization appears normal. The mutation corresponds to a strong loss of function in mbk-2, a conserved serine/threonine kinase. The microtubule-related defects are due to the postmeiotic persistence of MEI-1, a homologue of the microtubule-severing protein katanin. In addition, P-granule distribution is abnormal in mbk-2 mutants, consistent with genetic evidence that mbk-2 has other functions and with the requirement of mbk-2 activity at the one-cell stage. We propose that mbk-2 potentiates the degradation of MEI-1 and other proteins, possibly via direct phosphorylation.