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.

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.

Micro-computed tomography as a platform for exploring Drosophila development.

Understanding how events at the molecular and cellular scales contribute to tissue form and function is key to uncovering the mechanisms driving animal development, physiology and disease. Elucidating these mechanisms has been enhanced through the study of model organisms and the use of sophisticated genetic, biochemical and imaging tools. Here, we present an accessible method for non-invasive imaging of Drosophila melanogaster at high resolution using micro-computed tomography (µ-CT). We show how rapid processing of intact animals, at any developmental stage, provides precise quantitative assessment of tissue size and morphology, and permits analysis of inter-organ relationships. We then use µ-CT imaging to study growth defects in the Drosophila brain through the characterization of abnormal spindle (asp) and WD repeat domain 62 (Wdr62), orthologs of the two most commonly mutated genes in human microcephaly patients. Our work demonstrates the power of combining µ-CT with traditional genetic, cellular and developmental biology tools available in model organisms to address novel biological mechanisms that control animal development and disease.

A centrosomal scaffold shows some self-control.

The scaffolding protein AKAP350A is known to localize to the centrosome and the Golgi, but the molecular details of its function at the centrosome remain elusive. Using structure-function analyses, protein interaction assays, and super-resolution microscopy, Kolobova et al. now identify AKAP350A's specific location and protein partners at the centrosome. The authors further define an autoregulatory mechanism that likely controls AKAP350A's ability to nucleate microtubule growth.

Autoinhibition and relief mechanism for Polo-like kinase 4.

Polo-like kinase 4 (Plk4) is a master regulator of centriole duplication, and its hyperactivity induces centriole amplification. Homodimeric Plk4 has been shown to be ubiquitinated as a result of autophosphorylation, thus promoting its own degradation and preventing centriole amplification. Unlike other Plks, Plk4 contains three rather than two Polo box domains, and the function of its third Polo box (PB3) is unclear. Here, we performed a functional analysis of Plk4's structural domains. Like other Plks, Plk4 possesses a previously unidentified autoinhibitory mechanism mediated by a linker (L1) near the kinase domain. Thus, autoinhibition is a conserved feature of Plks. In the case of Plk4, autoinhibition is relieved after homodimerization and is accomplished by PB3 and by autophosphorylation of L1. In contrast, autophosphorylation of the second linker promotes separation of the Plk4 homodimer. Therefore, autoinhibition delays the multiple consequences of activation until Plk4 dimerizes. These findings reveal a complex mechanism of Plk4 regulation and activation which govern the process of centriole duplication.

Organelle asymmetry for proper fitness, function, and fate.

During cellular division, centrosomes are tasked with building the bipolar mitotic spindle, which partitions the cellular contents into two daughter cells. While every cell will receive an equal complement of chromosomes, not every organelle is symmetrically passaged to the two progeny in many cell types. In this review, we highlight the conservation of nonrandom centrosome segregation in asymmetrically dividing stem cells, and we discuss how the asymmetric function of centrosomes could mediate nonrandom segregation of organelles and mRNA. We propose that such a mechanism is critical for insuring proper cell fitness, function, and fate.

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 Proximal Centriole-Like Structure Anchors the Centriole to the Sperm Nucleus.

Proper connection between the sperm head and tail is critical for sperm motility and fertilization. The link between the head and tail is mediated by the Head-Tail Coupling Apparatus (HTCA), which secures the axoneme (tail) to the nucleus (head). However, the molecular architecture of the HTCA is not well understood. Here, we use Drosophila to create a high-resolution map of proteins and structures at the HTCA throughout spermiogenesis. Using structured illumination microscopy, we demonstrate that key HTCA proteins Spag4 and Yuri form a 'Centriole Cap' that surrounds the centriole (or Basal Body) as it is inserted, or embedded into the surface of the nucleus. As development progresses, the centriole is laterally displaces to the side of the nucleus, during which time the HTCA expands under the nucleus, forming what we term the 'Nuclear Shelf.' We next show that the proximal centriole-like (PCL) structure is positioned under the Nuclear Shelf and functions as a critical stabilizer of the centriole-nuclear attachment. Together, our data indicate that the HTCA is complex, multi-point attachment site that simultaneously engages the PCL, the centriole, and the nucleus to ensure proper head-tail connection during late-stage spermiogenesis.

Spd-2 gene duplication reveals cell-type-specific pericentriolar material regulation.

Centrosomes are multi-protein organelles that function as microtubule (MT) organizing centers (MTOCs), ensuring spindle formation and chromosome segregation during cell division.1,2,3 Centrosome structure includes core centrioles that recruit pericentriolar material (PCM) that anchors γ-tubulin to nucleate MTs.1,2 In Drosophila melanogaster, PCM organization depends on proper regulation of proteins like Spd-2, which dynamically localizes to centrosomes and is required for PCM, γ-tubulin, and MTOC activity in brain neuroblast (NB) mitosis and male spermatocyte (SC) meiosis.4,5,6,7,8 Some cells have distinct requirements for MTOC activity due to differences in characteristics like cell size9,10 or whether they are mitotic or meiotic.11,12 How centrosome proteins achieve cell-type-specific functional differences is poorly understood. Previous work identified alternative splicing13 and binding partners14 as contributors to cell-type-specific differences in centrosome function. Gene duplication, which can generate paralogs with specialized functions,15,16 is also implicated in centrosome gene evolution,17 including cell-type-specific centrosome genes.18,19 To gain insight into cell-type-specific differences in centrosome protein function and regulation, we investigated a duplication of Spd-2 in Drosophila willistoni, which has Spd-2A (ancestral) and Spd-2B (derived). We find that Spd-2A functions in NB mitosis, whereas Spd-2B functions in SC meiosis. Ectopically expressed Spd-2B accumulates and functions in mitotic NBs, but ectopically expressed Spd-2A failed to accumulate in meiotic SCs, suggesting cell-type-specific differences in translation or protein stability. We mapped this failure to accumulate and function in meiosis to the C-terminal tail domain of Spd-2A, revealing a novel regulatory mechanism that can potentially achieve differences in PCM function across cell types.

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.

Cep104 is a component of the centriole distal tip complex that regulates centriole growth and contributes to Drosophila spermiogenesis.

Proper centrosome number and function relies on the accurate assembly of centrioles, barrel-shaped structures that form the core duplicating elements of the organelle. The growth of centrioles is regulated in a cell cycle-dependent manner; while new daughter centrioles elongate during the S/G2/M phase, mature mother centrioles maintain their length throughout the cell cycle. Centriole length is controlled by the synchronized growth of the microtubules that ensheathe the centriole barrel. Although proteins exist that target the growing distal tips of centrioles, such as CP110 and Cep97, these proteins are generally thought to suppress centriolar microtubule growth, suggesting that distal tips may also contain unidentified counteracting factors that facilitate microtubule polymerization. Currently, a mechanistic understanding of how distal tip proteins balance microtubule growth and shrinkage to either promote daughter centriole elongation or maintain centriole length is lacking. Using a proximity-labeling screen in Drosophila cells, we identified Cep104 as a novel component of a group of evolutionarily conserved proteins that we collectively refer to as the distal tip complex (DTC). We found that Cep104 regulates centriole growth and promotes centriole elongation through its microtubule-binding TOG domain. Furthermore, analysis of Cep104 null flies revealed that Cep104 and Cep97 cooperate during spermiogenesis to align spermatids and coordinate individualization. Lastly, we mapped the complete DTC interactome and showed that Cep97 is the central scaffolding unit required to recruit DTC components to the distal tip of centrioles.

A role for a novel centrosome cycle in asymmetric cell division.

Tissue stem cells play a key role in tissue maintenance. Drosophila melanogaster central brain neuroblasts are excellent models for stem cell asymmetric division. Earlier work showed that their mitotic spindle orientation is established before spindle formation. We investigated the mechanism by which this occurs, revealing a novel centrosome cycle. In interphase, the two centrioles separate, but only one is active, retaining pericentriolar material and forming a "dominant centrosome." This centrosome acts as a microtubule organizing center (MTOC) and remains stationary, forming one pole of the future spindle. The second centriole is inactive and moves to the opposite side of the cell before being activated as a centrosome/MTOC. This is accompanied by asymmetric localization of Polo kinase, a key centrosome regulator. Disruption of centrosomes disrupts the high fidelity of asymmetric division. We propose a two-step mechanism to ensure faithful spindle positioning: the novel centrosome cycle produces a single interphase MTOC, coarsely aligning the spindle, and spindle-cortex interactions refine this alignment.

Pericentrin interacts with Kinesin-1 to drive centriole motility.

Centrosome positioning is essential for their function. Typically, centrosomes are transported to various cellular locations through the interaction of centrosomal microtubules (MTs) with motor proteins anchored at the cortex or the nuclear surface. However, it remains unknown how centrioles migrate in cellular contexts in which they do not nucleate MTs. Here, we demonstrate that during interphase, inactive centrioles move directly along the interphase MT network as Kinesin-1 cargo. We identify Pericentrin-Like-Protein (PLP) as a novel Kinesin-1 interacting molecule essential for centriole motility. In vitro assays show that PLP directly interacts with the cargo binding domain of Kinesin-1, allowing PLP to migrate on MTs. Binding assays using purified proteins revealed that relief of Kinesin-1 autoinhibition is critical for its interaction with PLP. Finally, our studies of neural stem cell asymmetric divisions in the Drosophila brain show that the PLP-Kinesin-1 interaction is essential for the timely separation of centrioles, the asymmetry of centrosome activity, and the age-dependent centrosome inheritance.

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.

Dynamic sex chromosome expression in Drosophila male germ cells.

Given their copy number differences and unique modes of inheritance, the evolved gene content and expression of sex chromosomes is unusual. In many organisms the X and Y chromosomes are inactivated in spermatocytes, possibly as a defense mechanism against insertions into unpaired chromatin. In addition to current sex chromosomes, Drosophila has a small gene-poor X-chromosome relic (4th) that re-acquired autosomal status. Here we use single cell RNA-Seq on fly larvae to demonstrate that the single X and pair of 4th chromosomes are specifically inactivated in primary spermatocytes, based on measuring all genes or a set of broadly expressed genes in testis we identified. In contrast, genes on the single Y chromosome become maximally active in primary spermatocytes. Reduced X transcript levels are due to failed activation of RNA-Polymerase-II by phosphorylation of Serine 2 and 5.

Centrosome fragments and microtubules are transported asymmetrically away from division plane in anaphase.

Spinning disc confocal microscopy of LLCPK1 cells expressing GFP-tubulin was used to demonstrate that microtubules (MTs) rapidly elongate to the cell cortex after anaphase onset. Concurrently, individual MTs are released from the centrosome and the centrosome fragments into clusters of MTs. Using cells expressing photoactivatable GFP-tubulin to mark centrosomal MT minus ends, a sevenfold increase in MT release in anaphase is documented as compared with metaphase. Transport of both individually released MTs and clusters of MTs is directionally biased: motion is directed away from the equatorial region. Clusters of MTs retain centrosomal components at their focus and the capacity to nucleate MTs. Injection of mRNA encoding nondegradable cyclin B blocked centrosome fragmentation and the stimulation of MT release in anaphase despite allowing anaphase-like chromosome segregation. Biased MT release may provide a mechanism for MT-dependent positioning of components necessary for specifying the site of contractile ring formation.

Sperm Head-Tail Linkage Requires Restriction of Pericentriolar Material to the Proximal Centriole End.

The centriole, or basal body, is the center of attachment between the sperm head and tail. While the distal end of the centriole templates the cilia, the proximal end associates with the nucleus. Using Drosophila, we identify a centriole-centric mechanism that ensures proper proximal end docking to the nucleus. This mechanism relies on the restriction of pericentrin-like protein (PLP) and the pericentriolar material (PCM) to the proximal end of the centriole. PLP is restricted proximally by limiting its mRNA and protein to the earliest stages of centriole elongation. Ectopic positioning of PLP to more distal portions of the centriole is sufficient to redistribute PCM and microtubules along the entire centriole length. This results in erroneous, lateral centriole docking to the nucleus, leading to spermatid decapitation as a result of a failure to form a stable head-tail linkage.

A molecular mechanism for the procentriole recruitment of Ana2.

During centriole duplication, a preprocentriole forms at a single site on the mother centriole through a process that includes the hierarchical recruitment of a conserved set of proteins, including the Polo-like kinase 4 (Plk4), Ana2/STIL, and the cartwheel protein Sas6. Ana2/STIL is critical for procentriole assembly, and its recruitment is controlled by the kinase activity of Plk4, but how this works remains poorly understood. A structural motif called the G-box in the centriole outer wall protein Sas4 interacts with a short region in the N terminus of Ana2/STIL. Here, we show that binding of Ana2 to the Sas4 G-box enables hyperphosphorylation of the Ana2 N terminus by Plk4. Hyperphosphorylation increases the affinity of the Ana2-G-box interaction, and, consequently, promotes the accumulation of Ana2 at the procentriole to induce daughter centriole formation.

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.