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Jordan W Raff

Publications and source records attributed to Jordan W Raff.

10 recordsLinked to original sources
PubMed2006

Flies without centrioles.

Centrioles and centrosomes have an important role in animal cell organization, but it is uncertain to what extent they are essential for animal development. The Drosophila protein DSas-4 is related to the human microcephaly protein CenpJ and the C. elegans centriolar protein Sas-4. We show that DSas-4 is essential for centriole replication in flies. DSas-4 mutants start to lose centrioles during embryonic development, and, by third-instar larval stages, no centrioles or centrosomes are detectable. Mitotic spindle assembly is slow in mutant cells, and approximately 30% of the asymmetric divisions of larval neuroblasts are abnormal. Nevertheless, mutant flies develop with near normal timing into morphologically normal adults. These flies, however, have no cilia or flagella and die shortly after birth because their sensory neurons lack cilia. Thus, centrioles are essential for the formation of centrosomes, cilia, and flagella, but, remarkably, they are not essential for most aspects of Drosophila development.

Animals↗
PubMed2005

Aurora A activates D-TACC-Msps complexes exclusively at centrosomes to stabilize centrosomal microtubules.

Centrosomes are the dominant sites of microtubule (MT) assembly during mitosis in animal cells, but it is unclear how this is achieved. Transforming acidic coiled coil (TACC) proteins stabilize MTs during mitosis by recruiting Minispindles (Msps)/XMAP215 proteins to centrosomes. TACC proteins can be phosphorylated in vitro by Aurora A kinases, but the significance of this remains unclear. We show that Drosophila melanogaster TACC (D-TACC) is phosphorylated on Ser863 exclusively at centrosomes during mitosis in an Aurora A-dependent manner. In embryos expressing only a mutant form of D-TACC that cannot be phosphorylated on Ser863 (GFP-S863L), spindle MTs are partially destabilized, whereas astral MTs are dramatically destabilized. GFP-S863L is concentrated at centrosomes and recruits Msps there but cannot associate with the minus ends of MTs. We propose that the centrosomal phosphorylation of D-TACC on Ser863 allows D-TACC-Msps complexes to stabilize the minus ends of centrosome-associated MTs. This may explain why centrosomes are such dominant sites of MT assembly during mitosis.

Animals↗
PubMed2005

Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis.

Centrosomes act as sites of microtubule growth, but little is known about how the number and stability of microtubules emanating from a centrosome are controlled during the cell cycle. We studied the role of the TACC3-XMAP215 complex in this process by using purified proteins and Xenopus laevis egg extracts. We show that TACC3 forms a one-to-one complex with and enhances the microtubule-stabilizing activity of XMAP215 in vitro. TACC3 enhances the number of microtubules emanating from mitotic centrosomes, and its targeting to centrosomes is regulated by Aurora A-dependent phosphorylation. We propose that Aurora A regulation of TACC3 activity defines a centrosome-specific mechanism for regulation of microtubule polymerization in mitosis.

Animals↗
PubMed2004

The Drosophila pericentrin-like protein is essential for cilia/flagella function, but appears to be dispensable for mitosis.

Centrosomes consist of a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Proteins that contain a Pericentrin/AKAP450 centrosomal targeting (PACT) domain have been implicated in recruiting several proteins to the PCM. We show that the only PACT domain protein in Drosophila (the Drosophila pericentrin-like protein [D-PLP]) is associated with both the centrioles and the PCM, and is essential for the efficient centrosomal recruitment of all six PCM components that we tested. Surprisingly, however, all six PCM components are eventually recruited to centrosomes during mitosis in d-plp mutant cells, and mitosis is not dramatically perturbed. Although viable, d-plp mutant flies are severely uncoordinated, a phenotype usually associated with defects in mechanosensory neuron function. We show that the sensory cilia of these neurons are malformed and the neurons are nonfunctional in d-plp mutants. Moreover, the flagella in mutant sperm are nonmotile. Thus, D-PLP is essential for the formation of functional cilia and flagella in flies.

Animals↗
PubMed2003

The ch-TOG/XMAP215 protein is essential for spindle pole organization in human somatic cells.

The ch-TOG/XMAP215 family of proteins bind directly to microtubules and appear to play an essential role in stabilizing spindle microtubules. These proteins stabilize microtubules mainly by influencing microtubule plus-end dynamics, yet, in vivo, they are all strongly concentrated at spindle poles, where the minus ends of the microtubules are concentrated. In Drosophila embryos, the centrosomal protein D-TACC is required to efficiently recruit ch-TOG/Msps to centrosomes. In humans, ch-TOG and the three known TACC proteins have been implicated in cancer, but their functions are unknown. Here we extensively depleted TACC3 and ch-TOG from HeLa cells using RNA interference. In TACC3-depleted cells, spindles are well organized, but microtubules are partially destabilized and ch-TOG is no longer concentrated on spindle microtubules. In ch-TOG-depleted cells, relatively robust spindles form, but the spindles are highly disorganized. Thus, in human somatic cells, ch-TOG appears to play a major role in organizing spindle poles, and a more minor role in stabilizing spindle microtubules that is, at least in part, mediated via an interaction with TACC3.

Animals↗
PubMed2002

The dynamic localisation of the Drosophila APC/C: evidence for the existence of multiple complexes that perform distinct functions and are differentially localised.

In Drosophila cells, the destruction of cyclin B is spatially regulated. In cellularised embryos, cyclin B is initially degraded on the mitotic spindle and is then degraded in the cytoplasm. In syncytial embryos, only the spindle-associated cyclin B is degraded at the end of mitosis. The anaphase promoting complex/cyclosome (APC/C) targets cyclin B for destruction, but its subcellular localisation remains controversial. We constructed GFP fusions of two core APC/C subunits, Cdc16 and Cdc27. These fusion proteins were incorporated into the endogenous APC/C and were largely localised in the cytoplasm during interphase in living syncytial embryos. Both fusion proteins rapidly accumulated in the nucleus prior to nuclear envelope breakdown but only weakly associated with mitotic spindles throughout mitosis. Thus, the global activation of a spatially restricted APC/C cannot explain the spatially regulated destruction of cyclin B. Instead, different subpopulations of the APC/C must be activated at different times to degrade cyclin B. Surprisingly, we noticed that GFP-Cdc27 associated with mitotic chromosomes, whereas GFP-Cdc16 did not. Moreover, reducing the levels of Cdc16 or Cdc27 by >90% in tissue culture cells led to a transient mitotic arrest that was both biochemically and morphologically distinct. Taken together, our results raise the intriguing possibility that there could be multiple forms of the APC/C that are differentially localised and perform distinct functions.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2002

The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and time.

In Drosophila cells cyclin B is normally degraded in two phases: (a) destruction of the spindle-associated cyclin B initiates at centrosomes and spreads to the spindle equator; and (b) any remaining cytoplasmic cyclin B is degraded slightly later in mitosis. We show that the APC/C regulators Fizzy (Fzy)/Cdc20 and Fzy-related (Fzr)/Cdh1 bind to microtubules in vitro and associate with spindles in vivo. Fzy/Cdc20 is concentrated at kinetochores and centrosomes early in mitosis, whereas Fzr/Cdh1 is concentrated at centrosomes throughout the cell cycle. In syncytial embryos, only Fzy/Cdc20 is present, and only the spindle-associated cyclin B is degraded at the end of mitosis. A destruction box-mutated form of cyclin B (cyclin B triple-point mutant [CBTPM]-GFP) that cannot be targeted for destruction by Fzy/Cdc20, is no longer degraded on spindles in syncytial embryos. However, CBTPM-GFP can be targeted for destruction by Fzr/Cdh1. In cellularized embryos, which normally express Fzr/Cdh1, CBTPM-GFP is degraded throughout the cell but with slowed kinetics. These findings suggest that Fzy/Cdc20 is responsible for catalyzing the first phase of cyclin B destruction that occurs on the mitotic spindle, whereas Fzr/Cdh1 is responsible for catalyzing the second phase of cyclin B destruction that occurs throughout the cell. These observations have important implications for the mechanisms of the spindle checkpoint.

Animals↗
PubMed2002

Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules.

Disruption of the function of the A-type Aurora kinase of Drosophila by mutation or RNAi leads to a reduction in the length of astral microtubules in syncytial embryos, larval neuroblasts, and cultured S2 cells. In neuroblasts, it can also lead to loss of an organized centrosome and its associated aster from one of the spindle poles, whereas the centrosome at the other pole has multiple centrioles. When centrosomes are present at the poles of aurA mutants or aurA RNAi spindles, they retain many antigens but are missing the Drosophila counterpart of mammalian transforming acidic coiled coil (TACC) proteins, D-TACC. We show that a subpopulation of the total Aurora A is present in a complex with D-TACC, which is a substrate for the kinase. We propose that one of the functions of Aurora A kinase is to direct centrosomal organization such that D-TACC complexed to the MSPS/XMAP215 microtubule-associated protein may be recruited, and thus modulate the behavior of astral microtubules.

Adenosine Triphosphate↗
PubMed2002

Centrosomes and cancer: lessons from a TACC.

The recent discovery that many cancer cells have centrosomal abnormalities suggests a link between centrosomes and cancer. Members of the transforming acidic coiled-coil (TACC) family of proteins have been implicated in cancer and are concentrated at centrosomes, where they regulate microtubule stability. I discuss a model of how the TACC proteins might contribute to cancer. This model predicts that defects in TACC function can make important contributions to the development of cancer but are unlikely to be the primary cause of cancer. The model might also apply to several other centrosomal proteins that have been linked to cancer.

Animals↗