Positioning centrioles and centrosomes.

Centrosomes are the primary microtubule organizer in eukaryotic cells. In addition to shaping the intracellular microtubule network and the mitotic spindle, centrosomes are responsible for positioning cilia and flagella. To fulfill these diverse functions, centrosomes must be properly located within cells, which requires that they undergo intracellular transport. Importantly, centrosome mispositioning has been linked to ciliopathies, cancer, and infertility. The mechanisms by which centrosomes migrate are diverse and context dependent. In many cells, centrosomes move via indirect motor transport, whereby centrosomal microtubules engage anchored motor proteins that exert forces on those microtubules, resulting in centrosome movement. However, in some cases, centrosomes move via direct motor transport, whereby the centrosome or centriole functions as cargo that directly binds molecular motors which then walk on stationary microtubules. In this review, we summarize the mechanisms of centrosome motility and the consequences of centrosome mispositioning and identify key questions that remain to be addressed.

Using Drosophila to uncover the role of organismal physiology and the tumor microenvironment in cancer.

Cancer metastasis causes over 90% of cancer patient fatalities. Poor prognosis is determined by tumor type, the tumor microenvironment (TME), organ-specific biology, and animal physiology. While model organisms do not fully mimic the complexity of humans, many processes can be studied efficiently owing to the ease of genetic, developmental, and cell biology studies. For decades, Drosophila has been instrumental in identifying basic mechanisms controlling tumor growth and metastasis. The ability to generate clonal populations of distinct genotypes in otherwise wild-type animals makes Drosophila a powerful system to study tumor-host interactions at the local and global scales. This review discusses advancements in tumor biology, highlighting the strength of Drosophila for modeling TMEs and systemic responses in driving tumor progression and metastasis.

The E3 ligase Poe promotes Pericentrin degradation.

Centrosomes are essential parts of diverse cellular processes, and precise regulation of the levels of their constituent proteins is critical for their function. One such protein is Pericentrin (PCNT) in humans and Pericentrin-like protein (PLP) in Drosophila. Increased PCNT expression and its protein accumulation are linked to clinical conditions including cancer, mental disorders, and ciliopathies. However, the mechanisms by which PCNT levels are regulated remain underexplored. Our previous study demonstrated that PLP levels are sharply down-regulated during early spermatogenesis and this regulation is essential to spatially position PLP on the proximal end of centrioles. We hypothesized that the sharp drop in PLP protein was a result of rapid protein degradation during the male germ line premeiotic G2 phase. Here, we show that PLP is subject to ubiquitin-mediated degradation and identify multiple proteins that promote the reduction of PLP levels in spermatocytes, including the UBR box containing E3 ligase Poe (UBR4), which we show binds to PLP. Although protein sequences governing posttranslational regulation of PLP are not restricted to a single region of the protein, we identify a region that is required for Poe-mediated degradation. Experimentally stabilizing PLP, via internal PLP deletions or loss of Poe, leads to PLP accumulation in spermatocytes, its mispositioning along centrioles, and defects in centriole docking in spermatids.

Traip controls mushroom body size by suppressing mitotic defects.

Microcephaly is a failure to develop proper brain size and neuron number. Mutations in diverse genes are linked to microcephaly, including several with DNA damage repair (DDR) functions; however, it is not well understood how these DDR gene mutations limit brain size. One such gene is TRAIP, which has multiple functions in DDR. We characterized the Drosophila TRAIP homolog nopo, hereafter traip, and found that traip mutants (traip-) have a brain-specific defect in the mushroom body (MB). traip- MBs were smaller and contained fewer neurons, but no neurodegeneration, consistent with human primary microcephaly. Reduced neuron numbers in traip- were explained by premature loss of MB neuroblasts (MB-NBs), in part via caspase-dependent cell death. Many traip- MB-NBs had prominent chromosome bridges in anaphase, along with polyploidy, aneuploidy or micronuclei. Traip localization during mitosis is sufficient for MB development, suggesting that Traip can repair chromosome bridges during mitosis if necessary. Our results suggest that proper brain size is ensured by the recently described role for TRAIP in unloading stalled replication forks in mitosis, which suppresses DNA bridges and premature neural stem cell loss to promote proper neuron number.

Host autophagy mediates organ wasting and nutrient mobilization for tumor growth.

During tumor growth-when nutrient and anabolic demands are high-autophagy supports tumor metabolism and growth through lysosomal organelle turnover and nutrient recycling. Ras-driven tumors additionally invoke non-autonomous autophagy in the microenvironment to support tumor growth, in part through transfer of amino acids. Here we uncover a third critical role of autophagy in mediating systemic organ wasting and nutrient mobilization for tumor growth using a well-characterized malignant tumor model in Drosophila melanogaster. Micro-computed X-ray tomography and metabolic profiling reveal that RasV12 ; scrib-/- tumors grow 10-fold in volume, while systemic organ wasting unfolds with progressive muscle atrophy, loss of body mass, -motility, -feeding, and eventually death. Tissue wasting is found to be mediated by autophagy and results in host mobilization of amino acids and sugars into circulation. Natural abundance Carbon 13 tracing demonstrates that tumor biomass is increasingly derived from host tissues as a nutrient source as wasting progresses. We conclude that host autophagy mediates organ wasting and nutrient mobilization that is utilized for tumor growth.

Fascetto interacting protein ensures proper cytokinesis and ploidy.

Cell division is critical for development, organ growth, and tissue repair. The later stages of cell division include the formation of the microtubule (MT)-rich central spindle in anaphase, which is required to properly define the cell equator, guide the assembly of the acto-myosin contractile ring and ultimately ensure complete separation and isolation of the two daughter cells via abscission. Much is known about the molecular machinery that forms the central spindle, including proteins needed to generate the antiparallel overlapping interzonal MTs. One critical protein that has garnered great attention is the protein regulator of cytokinesis 1, or Fascetto (Feo) in Drosophila, which forms a homodimer to cross-link interzonal MTs, ensuring proper central spindle formation and cytokinesis. Here, we report on a new direct protein interactor and regulator of Feo we named Feo interacting protein (FIP). Loss of FIP results in a reduction in Feo localization, rapid disassembly of interzonal MTs, and several defects related to cytokinesis failure, including polyploidization of neural stem cells. Simultaneous reduction in Feo and FIP results in very large, tumorlike DNA-filled masses in the brain that contain hundreds of centrosomes. In aggregate, our data show that FIP acts directly on Feo to ensure fully accurate cell division.

Stu2 uses a 15-nm parallel coiled coil for kinetochore localization and concomitant regulation of the mitotic spindle.

XMAP215/Dis1 family proteins are potent microtubule polymerases, critical for mitotic spindle structure and dynamics. While microtubule polymerase activity is driven by an N-terminal tumor overexpressed gene (TOG) domain array, proper cellular localization is a requisite for full activity and is mediated by a C-terminal domain. Structural insight into the C-terminal domain's architecture and localization mechanism remain outstanding. We present the crystal structure of the Saccharomyces cerevisiae Stu2 C-terminal domain, revealing a 15-nm parallel homodimeric coiled coil. The parallel architecture of the coiled coil has mechanistic implications for the arrangement of the homodimer's N-terminal TOG domains during microtubule polymerization. The coiled coil has two spatially distinct conserved regions: CRI and CRII. Mutations in CRI and CRII perturb the distribution and localization of Stu2 along the mitotic spindle and yield defects in spindle morphology including increased frequencies of mispositioned and fragmented spindles. Collectively, these data highlight roles for the Stu2 dimerization domain as a scaffold for factor binding that optimally positions Stu2 on the mitotic spindle to promote proper spindle structure and dynamics.

Actin dynamics and competition for myosin monomer govern the sequential amplification of myosin filaments.

The cellular mechanisms governing non-muscle myosin II (NM2) filament assembly are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we characterized and quantified the sequential amplification of NM2 filaments within lamellae, wherein filaments emanating from single nucleation events continuously partition, forming filament clusters that populate large-scale actomyosin structures deeper in the cell. Individual partitioning events coincide spatially and temporally with the movements of diverging actin fibres, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibres to which they are bound. Finally, we showed that partition frequency and filament growth rate in the lamella depend on MLCK, and that MLCK is competing with centrally active ROCK for a limiting pool of monomer with which to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and spatio-temporal regulation of NM2 filament assembly in cells.

A novel GSK3-regulated APC:Axin interaction regulates Wnt signaling by driving a catalytic cycle of efficient ßcatenin destruction.

APC, a key negative regulator of Wnt signaling in development and oncogenesis, acts in the destruction complex with the scaffold Axin and the kinases GSK3 and CK1 to target ßcatenin for destruction. Despite 20 years of research, APC's mechanistic function remains mysterious. We used FRAP, super-resolution microscopy, functional tests in mammalian cells and flies, and other approaches to define APC's mechanistic role in the active destruction complex when Wnt signaling is off. Our data suggest APC plays two roles: (1) APC promotes efficient Axin multimerization through one known and one novel APC:Axin interaction site, and (2) GSK3 acts through APC motifs R2 and B to regulate APC:Axin interactions, promoting high-throughput of ßcatenin to destruction. We propose a new dynamic model of how the destruction complex regulates Wnt signaling and how this goes wrong in cancer, providing insights into how this multiprotein signaling complex is assembled and functions via multivalent interactions.

Phosphoregulation of STIM1 leads to exclusion of the endoplasmic reticulum from the mitotic spindle.

The endoplasmic reticulum (ER) undergoes significant reorganization between interphase and mitosis, but the underlying mechanisms are unknown. Stromal interaction molecule 1 (STIM1) is an ER Ca(2+) sensor that activates store-operated Ca(2+) entry (SOCE) and also functions in ER morphogenesis through its interaction with the microtubule +TIP protein end binding 1 (EB1). We previously demonstrated that phosphorylation of STIM1 during mitosis suppresses SOCE. We now show that STIM1 phosphorylation is a major regulatory mechanism that excludes ER from the mitotic spindle. In mitotic HeLa cells, the ER forms concentric sheets largely excluded from the mitotic spindle. We show that STIM1 dissociates from EB1 in mitosis and localizes to the concentric ER sheets. However, a nonphosphorylatable STIM1 mutant (STIM1(10A)) colocalized extensively with EB1 and drove ER mislocalization by pulling ER tubules into the spindle. This effect was rescued by mutating the EB1 interaction site of STIM1(10A), demonstrating that aberrant association of STIM1(10A) with EB1 is responsible for the ER mislocalization. A STIM1 phosphomimetic exhibited significantly impaired +TIP tracking in interphase but was ineffective at inhibiting SOCE, suggesting different mechanisms of regulation of these two STIM1 functions by phosphorylation. Thus, ER spindle exclusion and ER-dependent Ca(2+) signaling during mitosis require multimodal STIM1 regulation by phosphorylation.

Centrosome function: sometimes less is more.

Tight regulation of centrosome duplication is critical to ensure that centrosome number doubles once and only once per cell cycle. Superimposed onto this centrosome duplication cycle is a functional centrosome cycle in which they alternate between phases of quiescence and robust microtubule (MT) nucleation and MT-anchoring activities. In vertebrate cycling cells, interphase centrioles accumulate less pericentriolar material (PCM), reducing their MT nucleation capacity. In mitosis, centrosomes mature, accumulating more PCM to increase their nucleation and anchoring capacities to form robust MT asters. Interestingly, functional cycles of centrosomes can be altered to suit the cell's needs. Some interphase centrosomes function as a microtubule-organizing center by increasing their ability to anchor MTs to form centrosomal radial arrays. Other interphase centrosomes maintain their MT nucleation capacity but reduce/eliminate their MT-anchoring capacity. Recent work demonstrates that Drosophila cells take this to the extreme, whereby centrioles lose all detectable PCM during interphase, offering an explanation as to how centrosome-deficient flies develop to adulthood. Drosophila stem cells further modify the functional cycle by differentially regulating their two centrioles - a situation that seems important for stem cell asymmetric divisions, as misregulation of centrosome duplication in stem/progenitor cells can promote tumor formation. Here, we review recent findings that describe variations in the functional cycle of centrosomes.

Putting the model to the test: are APC proteins essential for neuronal polarity, axon outgrowth, and axon targeting?

The highly polarized architecture of neurons is important for their function. Experimental data based on dominant-negative approaches suggest that the tumor suppressor adenomatous polyposis coli (APC), a regulator of Wnt signaling and the cytoskeleton, regulates polarity of neuroectodermal precursors and neurons, helping specify one neurite as the axon, promoting its outgrowth, and guiding axon pathfinding. However, such dominant-negative approaches might affect processes in which APC is not essential. We completely removed both APCs from Drosophila melanogaster larval neural precursors and neurons, testing whether APCs play universal roles in neuronal polarity. Surprisingly, APCs are not essential for asymmetric cell division or the stereotyped division axis of central brain (CB) neuroblasts, although they do affect cell cycle progression and spindle architecture. Likewise, CB, lobular plug, and mushroom body neurons do not require APCs for polarization, axon outgrowth, or, in the latter two cases, axon targeting. These data suggest that proposed cytoskeletal roles for APCs in mammals should be reassessed using loss of function tools.

Original CIN: reviewing roles for APC in chromosome instability.

You may have seen the bumper sticker "Eve was framed." Thousands of years of being blamed for original sin and still many wonder, where's the evidence? Today, the tumor suppressor adenomatous polyposis coli (APC) may have the same complaint about accusations of a different type of CIN, chromosome instability. A series of recent papers, including three in this journal, propose that loss of APC function plays an important role in the CIN seen in many colon cancer cells. However, a closer look reveals a complex story that raises more questions than answers.

A multicomponent assembly pathway contributes to the formation of acentrosomal microtubule arrays in interphase Drosophila cells.

In animal cells, centrosomes nucleate microtubules that form polarized arrays to organize the cytoplasm. Drosophila presents an interesting paradox however, as centrosome-deficient mutant animals develop into viable adults. To understand this discrepancy, we analyzed behaviors of centrosomes and microtubules in Drosophila cells, in culture and in vivo, using a combination of live-cell imaging, electron microscopy, and RNAi. The canonical model of the cycle of centrosome function in animal cells states that centrosomes act as microtubule-organizing centers throughout the cell cycle. Unexpectedly, we found that many Drosophila cell-types display an altered cycle, in which functional centrosomes are only present during cell division. On mitotic exit, centrosomes disassemble producing interphase cells containing centrioles that lack microtubule-nucleating activity. Furthermore, steady-state interphase microtubule levels are not changed by codepleting both gamma-tubulins. However, gamma-tubulin RNAi delays microtubule regrowth after depolymerization, suggesting that it may function partially redundantly with another pathway. Therefore, we examined additional microtubule nucleating factors and found that Mini-spindles, CLIP-190, EB1, or dynein RNAi also delayed microtubule regrowth; surprisingly, this was not further prolonged when we codepleted gamma-tubulins. Taken together, these results modify our view of the cycle of centrosome function and reveal a multi-component acentrosomal microtubule assembly pathway to establish interphase microtubule arrays in Drosophila.

SV40 VP2 and VP3 insertion into ER membranes is controlled by the capsid protein VP1: implications for DNA translocation out of the ER.

Nonenveloped viruses such as Simian Virus 40 (SV40) exploit established cellular pathways for internalization and transport to their site of penetration. By analyzing mutant SV40 genomes that do not express VP2 or VP3, we found that these structural proteins perform essential functions that are regulated by VP1. VP2 significantly enhanced SV40 particle association with the host cell, while VP3 functioned downstream. VP2 and VP3 both integrated posttranslationally into the endoplasmic reticulum (ER) membrane. Association with VP1 pentamers prevented their ER membrane integration, indicating that VP1 controls the function of VP2 and VP3 by directing their localization between the particle and the ER membrane. These findings suggest a model in which VP2 aids in cell binding. After capsid disassembly within the ER lumen, VP3, and perhaps VP2, oligomerizes and integrates into the ER membrane, potentially creating a viroporin that aids in viral DNA transport out of the ER.

Simian virus 40 late proteins possess lytic properties that render them capable of permeabilizing cellular membranes.

Many nonenveloped viruses have evolved an infectious cycle that culminates in the lysis or permeabilization of the host to enable viral release. How these viruses initiate the lytic event is largely unknown. Here, we demonstrated that the simian virus 40 progeny accumulated at the nuclear envelope prior to the permeabilization of the nuclear, endoplasmic reticulum, and plasma membranes at a time which corresponded with the release of the progeny. The permeabilization of these cellular membranes temporally correlated with late protein expression and was not observed upon the inhibition of their synthesis. To address whether one or more of the late proteins possessed an inherent capacity to induce membrane permeabilization, we examined the permeability of Escherichia coli that separately expressed the late proteins. VP2 and VP3, but not VP1, caused the permeabilization of bacterial membranes. Additionally, VP3 expression resulted in bacterial cell lysis. These findings demonstrate that VP3 possesses an inherent lytic property that is independent of eukaryotic signaling or cell death pathways.

Reorganization of the microtubule array in prophase/prometaphase requires cytoplasmic dynein-dependent microtubule transport.

When mammalian somatic cells enter mitosis, a fundamental reorganization of the Mt cytoskeleton occurs that is characterized by the loss of the extensive interphase Mt array and the formation of a bipolar mitotic spindle. Microtubules in cells stably expressing GFP-alpha-tubulin were directly observed from prophase to just after nuclear envelope breakdown (NEBD) in early prometaphase. Our results demonstrate a transient stimulation of individual Mt dynamic turnover and the formation and inward motion of microtubule bundles in these cells. Motion of microtubule bundles was inhibited after antibody-mediated inhibition of cytoplasmic dynein/dynactin, but was not inhibited after inhibition of the kinesin-related motor Eg5 or myosin II. In metaphase cells, assembly of small foci of Mts was detected at sites distant from the spindle; these Mts were also moved inward. We propose that cytoplasmic dynein-dependent inward motion of Mts functions to remove Mts from the cytoplasm at prophase and from the peripheral cytoplasm through metaphase. The data demonstrate that dynamic astral Mts search the cytoplasm for other Mts, as well as chromosomes, in mitotic cells.

Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma.

Cellular microtubules, polymers of tubulin, alternate relentlessly between phases of growth and shortening. We now show that noscapine, a tubulin-binding agent, increases the time that cellular microtubules spend idle in a paused state. As a result, most mammalian cell types observed arrest in mitosis in the presence of noscapine. We demonstrate that noscapine-treated murine melanoma B16LS9 cells do not arrest in mitosis but rather become polyploid followed by cell death, whereas primary melanocytes reversibly arrest in mitosis and resume a normal cell cycle after noscapine removal. Furthermore, in a syngeneic murine model of established s.c. melanoma, noscapine treatment resulted in an 85% inhibition of tumor volume on day 17 when delivered by gavage compared with untreated animals (P