We determined the distribution of 11 different transposable elements on Drosophila melanogaster mitotic chromosomes by using high-resolution fluorescent in situ hybridization (FISH) coupled with charge-coupled device camera analysis. Nine of these transposable elements (copia, gypsy, mdg-1, blood, Doc, I, F, G, and Bari-]) are preferentially clustered into one or more discrete heterochromatic regions in chromosomes of the Oregon-R laboratory stoclk Moreover, FISH analysis of geographically distant strains revealed that the locations of these heterochromatic transposable element clusters are highly conserved. The P and hobo elements, which are likely to have invaded the D. melanogaster genome at the beginning of this century, are absent from Oregon-R heterochromatin but clearly exhibit heterochromatic clusters in certain natural populations. Together these data indicate that transposable elements are major structural components of Drosophila heterochromatin, and they change the current views on the role of transposable elements in host genome evolution.

Spatial control is a key issue in cell division. The Ran GTPase regulates several fundamental processes for cell life, largely acting through importin molecules. The best understood of these is protein import through the nuclear envelope in interphase, but roles in mitotic spindle assembly are also established. In mammalian cells, in which centrosomes are major spindle organizers, a link is emerging between the Ran network, centrosomes and spindle poles. Here, we show that, after nuclear envelope breakdown, importin β is transported to the spindle poles in mammalian cells. This localization is temporally regulated from prometaphase until anaphase, when importin β dissociates from poles and is recruited back around reforming nuclei. Importin β sediments with mitotic microtubules in vitro and its accumulation at poles requires microtubule integrity and dynamics in vivo. Furthermore, RNA interference-dependent inactivation of TPX2, the major Ran-dependent spindle organizer, abolishes importin β accumulation at poles. Importin β has a functional role in spindle pole organization, because overexpression yields mitotic spindles with abnormal, fragmented poles. Coexpression of TPX2 with importin β mitigates these abnormalities. Together, these results indicate that the balance between importins and spindle regulators of the TPX2 type is crucial for spindle formation. Targeting of TPX2/importin-β complexes to poles is a key aspect in Ran-dependent control of the mitotic apparatus in mammalian cells.

Although membrane phospholipid phosphatidylinositol-4,5bisphosphate (PIP2) plays a key role as signaling intermediate and coordinator of actin dynamics and vesicle trafficking, it remains completely unknown its involvement in the activation of cytolytic machinery. By live confocal imaging of primary human natural killer (NK) cells expressing the chimeric protein GFP-PH, we observed, during effector-target cell interaction, the consumption of a preexisting PIP2 pool, which is critically required for the activation of cytolytic machinery. We identified type I phosphatidylinositol-4-phosphate-5-kinase (PI5KI) ␣ and ␥ isoforms as the enzymes responsible for PIP2 synthesis in NK cells. By hRNA-driven gene silencing, we observed that both enzymes are required for the proper activation of NK cytotoxicity and for inositol-1,4,5-trisphosphate (IP3) generation on receptor stimulation. In an attempt to elucidate the specific step controlled by PI5KIs, we found that lytic granule secretion but not polarization resulted in impaired PI5KI␣-and PI5KI␥-silenced cells. Our findings delineate a novel mechanism implicating PI5KI␣ and PI5KI␥ isoforms in the synthesis of PIP2 pools critically required for IP3-dependent Ca 2؉ IntroductionNatural killer (NK) cells and cytotoxic T lymphocytes are critical effectors in the defense against tumor and viral infections 1 ; they exert cytotoxic function through the polarized secretion of granules containing proteolytic molecules, such as perforin and granzymes. This process involves several steps, including the formation of a cytolytic synapse between cytolytic effector and target cell, the rapid reorientation of the microtubule-organizing center along with lytic granules toward the target contact area followed by granule docking and fusion at specialized secretory domains within the cytolytic synapse. 2,3 Several structurally distinct receptors have been implicated in the activation of NK-cell cytolytic machinery: when cross-linked by the corresponding ligands on target cell, they trigger multiple and intersecting signaling pathways responsible for functional activation. 4 A vast array of activating NK receptors belonging to different families are coupled to the lipid modifying enzymes phosphatidylinositol3-kinase (PI3K) and phospholipase C␥ (PLC␥), which provide signals critically required for the activation of the cytolytic machinery [5][6][7][8] ; notably, both enzymes use the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) as common substrate. 9 Besides its role as signaling intermediate, PIP2 also acts as critical regulator of various cellular processes, including actin remodeling, membrane and vesicle trafficking, adhesion, and ion transport. 10,11 Surprisingly, the role of PIP2 and its regulatory mechanisms in lymphocyte-mediated cytotoxicity remain completely undefined.The main cellular source of PIP2 are type I phosphatidylinositol-4-phosphate-5-kinase (PI5KI) family members which phosphorylate PI4P on the D5 position of the inositol ring. Three major PI5KI is...

In Drosophila, the Polycomb group and trithorax group proteins play a critical role in controlling the expression states of homeotic gene complexes during development. The common view is that these two classes of proteins bind to the homeotic complexes and regulate transcription at the level of chromatin. In the present work, we tested the involvement of both groups in mitotic heterochromatin formation in Drosophila. Using specific antibodies, we show that some of the tested Pc-G proteins are present in heterochromatin, while all the tested trx-G proteins localize to specific regions of heterochromatin in both mitotic chromosomes and interphase nuclei. We also observed that mutations in trx-G genes are recessive enhancers of position-effect variegation and are able to repress the transcription of heterochromatic genes. These results strongly suggest that trx-G proteins, along with some Pc-G proteins, play an active role in heterochromatin formation in Drosophila.

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