Liquid-liquid phase separation has been shown to underlie the formation and disassembly of membraneless organelles in cells, but the cellular mechanisms that control this phenomenon are poorly understood. A prominent example of regulated and reversible segregation of liquid phases may occur during mitosis, when membraneless organelles disappear upon nuclear-envelope breakdown and reappear as mitosis is completed. Here we show that the dual-specificity kinase DYRK3 acts as a central dissolvase of several types of membraneless organelle during mitosis. DYRK3 kinase activity is essential to prevent the unmixing of the mitotic cytoplasm into aberrant liquid-like hybrid organelles and the over-nucleation of spindle bodies. Our work supports a mechanism in which the dilution of phase-separating proteins during nuclear-envelope breakdown and the DYRK3-dependent degree of their solubility combine to allow cells to dissolve and condense several membraneless organelles during mitosis.

Stable isotope labeling by amino acids in cell culture (SILAC) is widely used to quantify protein abundance in tissue culture cells. Until now, the only multicellular organism completely labeled at the amino acid level was the laboratory mouse. The fruit fly Drosophila melanogaster is one of the most widely used small animal models in biology. Here, we show that feeding flies with SILAC-labeled yeast leads to almost complete labeling in the first filial generation. We used these "SILAC flies" to investigate sexual dimorphism of protein abundance in D. melanogaster. Quantitative proteome comparison of adult male and female flies revealed distinct biological processes specific for each sex. Using a tudor mutant that is defective for germ cell generation allowed us to differentiate between sex-specific protein expression in the germ line and somatic tissue. We identified many proteins with known sexspecific expression bias. In addition, several new proteins with a potential role in sexual dimorphism were identified. Collectively, our data show that the SILAC fly can be used to accurately quantify protein abundance in vivo. The approach is simple, fast, and cost-effective, making SILAC flies an attractive model system for the emerging field of in vivo quantitative proteomics. Molecular & Cellular Proteomics 9:2173-2183, 2010.Mass spectrometry-based quantitative proteomics has emerged as a highly successful approach to study biological processes in health and disease (1-3). Most studies have so far been limited to in vitro systems such as cell culture models. Although tremendously useful, these models cannot appropriately reflect relevant regulatory mechanisms of multicellular eukaryotes in vivo. This is particularly relevant for complex processes involving interactions between different cell types such as differentiation and development (4).Relative changes in protein abundance are most accurately measured by comparing the natural form of a peptide with its stable isotope-labeled analog. Several different approaches enable stable isotope labeling of peptides either by chemical reactions or metabolic incorporation of the label (5, 6). Metabolic labeling has several advantages such as high labeling efficiency and intrinsically higher precision. For example, metabolically labeled samples can be combined before further processing steps so that protein quantification is not affected by differences in sample preparation. Labeling of organisms with stable isotope tracers was pioneered by Rudolf Schoenheimer 75 years ago (7,8). Since then, several model organisms ranging from prokaryotes to mammals have been labeled metabolically (for an excellent review, see Ref. 9). For example, Caenorhabditis elegans and Drosophila melanogaster have successfully been labeled with 15 N (10), and 15 N-labeled flies were recently used to study maternal-tozygotic transition (11) and seminal fluid proteins (sfps) 1 transferred at mating (12).15 N has also been used to label entire rats, particularly for quantitative brain proteomics (13,14). ...

Centrioles are core structural elements of both centrosomes and cilia. Although cytoplasmic granules called centriolar satellites have been observed around these structures, lack of a comprehensive inventory of satellite proteins impedes our understanding of their ancestry. To address this, we performed mass spectrometry (MS)‐based proteome profiling of centriolar satellites obtained by affinity purification of their key constituent, PCM1, from sucrose gradient fractions. We defined an interactome consisting of 223 proteins, which showed striking enrichment in centrosome components. The proteome also contained new structural and regulatory factors with roles in ciliogenesis. Quantitative MS on whole‐cell and centriolar satellite proteomes of acentriolar cells was performed to reveal dependencies of satellite composition on intact centrosomes. Although most components remained associated with PCM1 in acentriolar cells, reduced cytoplasmic and satellite levels were observed for a subset of centrosomal proteins. These results demonstrate that centriolar satellites and centrosomes form independently but share a substantial fraction of their proteomes. Dynamic exchange of proteins between these organelles could facilitate their adaptation to changing cellular environments during development, stress response and tissue homeostasis.