The AID2 system offers a potent tool for rapid, reversible, or sustained degradation of essential proteins in live mice.

Studying essential genes required for dynamic processes in live mice is challenging as genetic perturbations are irreversible and limited by slow protein depletion kinetics. The first-generation auxin-inducible-degron (AID) system is a powerful tool for analyzing inducible protein loss in cultured cells. However, auxin administration is toxic to mice, preventing its long-term use in animals. Here, we use an optimized second-generation AID system to achieve the conditional and reversible loss of the essential centrosomal protein CEP192 in live mice. We show that the auxin derivative 5-Ph-IAA is well tolerated over two weeks and drives near-complete CEP192-mAID degradation in less than one hour in vivo. Prolonged CEP192 loss led to cell division failure and cell death in proliferative tissues. Thus, the second-generation AID system is well suited for rapid and/or sustained protein depletion in live mice, offering a valuable new tool for interrogating protein function in vivo.

Centriole structural integrity defects are a crucial feature of Hydrolethalus Syndrome.

Hydrolethalus Syndrome (HLS) is a lethal, autosomal recessive ciliopathy caused by the mutation of the conserved centriole protein HYLS1. However, how HYLS1 facilitates the centriole-based templating of cilia is poorly understood. Here, we show that mice harboring the HYLS1 disease mutation die shortly after birth and exhibit developmental defects that recapitulate several manifestations of the human disease. These phenotypes arise from tissue-specific defects in cilia assembly and function caused by a loss of centriole integrity. We show that HYLS1 is recruited to the centriole by CEP120 and functions to recruit centriole inner scaffold proteins that stabilize the centriolar microtubule wall. The HLS mutation disrupts the interaction of HYLS1 with CEP120 leading to HYLS1 displacement and degeneration of the centriole distal end. We propose that tissue-specific defects in centriole integrity caused by the HYLS1 mutation prevent ciliogenesis and drive HLS phenotypes.

Trisomy 21 induces pericentrosomal crowding delaying primary ciliogenesis and mouse cerebellar development.

Trisomy 21, the genetic cause of Down syndrome, disrupts primary cilia formation and function, in part through elevated Pericentrin, a centrosome protein encoded on chromosome 21. Yet how trisomy 21 and elevated Pericentrin disrupt cilia-related molecules and pathways, and the in vivo phenotypic relevance remain unclear. Utilizing ciliogenesis time course experiments combined with light microscopy and electron tomography, we reveal that chromosome 21 polyploidy elevates Pericentrin and microtubules away from the centrosome that corral MyosinVA and EHD1, delaying ciliary membrane delivery and mother centriole uncapping essential for ciliogenesis. If given enough time, trisomy 21 cells eventually ciliate, but these ciliated cells demonstrate persistent trafficking defects that reduce transition zone protein localization and decrease sonic hedgehog signaling in direct anticorrelation with Pericentrin levels. Consistent with cultured trisomy 21 cells, a mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers at P4. Our work reveals that elevated Pericentrin from trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding mechanism results in signaling deficiencies consistent with the neurological phenotypes found in individuals with Down syndrome.

Human cells typically have 23 pairs of structures known as chromosomes. Each chromosome contains a unique set of genes which provide the instructions needed to make proteins and other essential molecules found in the body. Individuals with Down syndrome have an extra copy of chromosome 21. This genetic alteration is known as trisomy 21 and affects many different organs in the body, leading to various medical conditions including intellectual disability, heart defects, and immune deficiencies. A recent study showed that cells from individuals with Down syndrome had defects in forming primary cilia ­ structures on the surface of cells which work as signaling hubs to control how cells grow and develop. These cilia defects were in large part due to excess levels of a protein known as Pericentrin, which is encoded by a gene found on chromosome 21. But it is unclear how Pericentrin disrupts cilia assembly, and how this may contribute to the medical conditions observed in individuals with Down syndrome. To address these questions, Jewett et al. studied human cells that had been engineered to have trisomy 21. The experiments found that trisomy 21 led to higher levels of Pericentrin and altered the way molecules were organized at the sites where primary cilia form. This caused the components required to build and maintain the primary cilium to become trapped in the wrong locations. The trisomy 21 cells were eventually able to rearrange the molecules and build a primary cilium, but it took them twice as long as cells with 23 pairs of chromosomes and their primary cilium did not properly work. Further experiments were then conducted on mice that had been engineered to have an extra copy of a portion of genes on human chromosome 21, including the gene for Pericentrin. Jewett et al. found that these mice assembled cilia later and had defects in cilia signaling, similar to the human trisomy 21 cells. This resulted in mild abnormalities in brain development that were consistent with what occurs in individuals with Down syndrome. These findings suggest that the elevated levels of Pericentrin in trisomy 21 causes changes in cilia formation and function which, in turn, may alter how the mouse brain develops. Further studies will be required to find out whether defects in primary cilia may contribute to other medical conditions observed in individuals with Down syndrome.

PLK4 drives centriole amplification and apical surface area expansion in multiciliated cells.

Multiciliated cells (MCCs) are terminally differentiated epithelia that assemble multiple motile cilia used to promote fluid flow. To template these cilia, MCCs dramatically expand their centriole content during a process known as centriole amplification. In cycling cells, the master regulator of centriole assembly Polo-like kinase 4 (PLK4) is essential for centriole duplication; however recent work has questioned the role of PLK4 in centriole assembly in MCCs. To address this discrepancy, we created genetically engineered mouse models and demonstrated that both PLK4 protein and kinase activity are critical for centriole amplification in MCCs. Tracheal epithelial cells that fail centriole amplification accumulate large assemblies of centriole proteins and do not undergo apical surface area expansion. These results show that the initial stages of centriole assembly are conserved between cycling cells and MCCs and suggest that centriole amplification and surface area expansion are coordinated events.

Every day, we inhale thousands of viruses, bacteria and pollution particles. To protect against these threats, cells in our airways produce mucus that traps inhaled particles before they reach the lungs. This mucus then needs to be removed to prevent it from becoming a breeding ground for microbes that may cause a respiratory infection. This is the responsibility of cells covered in tiny hair-like structures called cilia that move together to propel the mucus-trapped particles out of the airways. These specialized cells can have up to 300 motile cilia on their surface, which grow from structures called centrioles that then anchor the cilia in place. Multiciliated cells are generated from precursor cells that only have two centrioles. Therefore, as these precursors develop, they must produce large numbers of centrioles, considerably more than other cells that only need a couple of extra centrioles during cell division. However, recent studies have questioned whether the precursors of multiciliated cells rely on the same regulatory proteins to produce centrioles as dividing cells. To help answer this question, LoMastro et al. created genetically engineered mice that lacked or had an inactive form of PLK4, a protein which controls centriole formation in all cell types lacking multiple cilia. This showed that multiciliated cells also need this protein to produce centrioles. LoMastro et al. also found that multiciliated cells became larger while building centrioles, suggesting that this amplification process helps control the cell's final size. Defects in motile cilia activity can lead to fluid build-up in the brain, respiratory infections and infertility. Unfortunately, these disorders are difficult to diagnose currently and there is no cure. The findings of LoMastro et al. further our understanding of how motile cilia are built and maintained, and may help future scientists to develop better diagnostic tools and treatments for patients.

Trisomy 21 increases microtubules and disrupts centriolar satellite localization.

Trisomy 21, the source of Down syndrome, causes a 0.5-fold protein increase of the chromosome 21-resident gene Pericentrin (PCNT) and reduces primary cilia formation and signaling. We investigate how PCNT imbalances disrupt cilia. Using isogenic RPE-1 cells with increased chromosome 21 dosage, we find PCNT accumulates around the centrosome as a cluster of enlarged cytoplasmic puncta that localize along microtubules (MTs) and at MT ends. Cytoplasmic PCNT puncta impact the density, stability, and localization of the MT trafficking network required for primary cilia. The PCNT puncta appear to sequester cargo peripheral to centrosomes in what we call pericentrosomal crowding. The centriolar satellite proteins PCM1, CEP131, and CEP290, important for ciliogenesis, accumulate at enlarged PCNT puncta in trisomy 21 cells. Reducing PCNT when chromosome 21 ploidy is elevated is sufficient to decrease PCNT puncta and pericentrosomal crowding, reestablish a normal density of MTs around the centrosome, and restore ciliogenesis to wild-type levels. A transient reduction in MTs also decreases pericentrosomal crowding and partially rescues ciliogenesis in trisomy 21 cells, indicating that increased PCNT leads to defects in the MT network deleterious to normal centriolar satellite distribution. We propose that chromosome 21 aneuploidy disrupts MT-dependent intracellular trafficking required for primary cilia.

Cancer biology: Messages in extracellular vesicles depend on centrosome number.

Extra centrosomes are linked to cancer-associated errors in cell division, metastasis and signaling. A new study reveals that centrosome amplification disrupts lysosome function, leading to the release of small extracellular vesicles and to invasive activity in pancreatic cells.

RAB19 Directs Cortical Remodeling and Membrane Growth for Primary Ciliogenesis.

Primary cilia are sensory organelles that utilize the compartmentalization of membrane and cytoplasm to communicate signaling events, and yet, how the formation of a cilium is coordinated with reorganization of the cortical membrane and cytoskeleton is unclear. Using polarized epithelia, we find that cortical actin clearing and apical membrane partitioning occur where the centrosome resides at the cell surface prior to ciliation. RAB19, a previously uncharacterized RAB, associates with the RAB-GAP TBC1D4 and the HOPS-tethering complex to coordinate cortical clearing and ciliary membrane growth, which is essential for ciliogenesis. This RAB19-directed pathway is not exclusive to polarized epithelia, as RAB19 loss in nonpolarized cell types blocks ciliogenesis with a docked ciliary vesicle. Remarkably, inhibiting actomyosin contractility can substitute for the function of the RAB19 complex and restore ciliogenesis in knockout cells. Together, this work provides a mechanistic understanding behind a cytoskeletal clearing and membrane partitioning step required for ciliogenesis.

The Rab11 effectors Fip5 and Fip1 regulate zebrafish intestinal development.

The Rab11 apical recycling endosome pathway is a well-established regulator of polarity and lumen formation; however, Rab11-vesicular trafficking also directs a diverse array of other cellular processes, raising the question of how Rab11 vesicles achieve specificity in space, time and content of cargo delivery. In part, this specificity is achieved through effector proteins, yet the role of Rab11 effector proteins in vivo remains vague. Here, we use CRISPR/Cas9 gene editing to study the role of the Rab11 effector Fip5 during zebrafish intestinal development. Zebrafish contain two paralogous genes, fip5a and fip5b, that are orthologs of human FIP5 We find that fip5a- and fip5b-mutant fish show phenotypes characteristic of microvillus inclusion disease, including microvilli defects and lysosomal accumulation. Single and double mutant analyses suggest that fip5a and fip5b function in parallel and regulate trafficking pathways required for assembly of keratin at the terminal web. Remarkably, in some genetic backgrounds, the absence of Fip5 triggers protein upregulation of a closely related family member, Fip1. This compensation mechanism occurs both during zebrafish intestinal development and in tissue culture models of lumenogenesis. In conclusion, our data implicate the Rab11 effectors Fip5 and Fip1 in a trafficking pathway required for apical microvilli formation.

Cell ratcheting through the Sbf RabGEF directs force balancing and stepped apical constriction.

During Drosophila melanogaster gastrulation, the invagination of the prospective mesoderm is driven by the pulsed constriction of apical surfaces. Here, we address the mechanisms by which the irreversibility of pulsed events is achieved while also permitting uniform epithelial behaviors to emerge. We use MSD-based analyses to identify contractile steps and find that when a trafficking pathway initiated by Sbf is disrupted, contractile steps become reversible. Sbf localizes to tubular, apical surfaces and associates with Rab35, where it promotes Rab GTP exchange. Interestingly, when Sbf/Rab35 function is compromised, the apical plasma membrane becomes deeply convoluted, and nonuniform cell behaviors begin to emerge. Consistent with this, Sbf/Rab35 appears to prefigure and organize the apical surface for efficient Myosin function. Finally, we show that Sbf/Rab35/CME directs the plasma membrane to Rab11 endosomes through a dynamic interaction with Rab5 endosomes. These results suggest that periodic ratcheting events shift excess membrane from cell apices into endosomal pathways to permit reshaping of actomyosin networks and the apical surface.

Vertex sliding drives intercalation by radial coupling of adhesion and actomyosin networks during Drosophila germband extension.

Oriented cell intercalation is an essential developmental process that shapes tissue morphologies through the directional insertion of cells between their neighbors. Previous research has focused on properties of cell-cell interfaces, while the function of tricellular vertices has remained unaddressed. Here, we identify a highly novel mechanism in which vertices demonstrate independent sliding behaviors along cell peripheries to produce the topological deformations responsible for intercalation. Through systematic analysis, we find that the motion of vertices connected by contracting interfaces is not physically coupled, but instead possess strong radial coupling. E-cadherin and Myosin II exist in previously unstudied populations at cell vertices and undergo oscillatory cycles of accumulation and dispersion that are coordinated with changes in cell area. Additionally, peak enrichment of vertex E-cadherin/Myosin II coincides with interface length stabilization. Our results suggest a model in which asymmetric radial force balance directs the progressive, ratcheted motion of individual vertices to drive intercalation.

Insane in the apical membrane: Trafficking events mediating apicobasal epithelial polarity during tube morphogenesis.

The creation of cellular tubes is one of the most vital developmental processes, resulting in the formation of most organ types. Cells have co-opted a number of different mechanisms for tube morphogenesis that vary among tissues and organisms; however, generation and maintenance of cell polarity is fundamental for successful lumenogenesis. Polarized membrane transport has emerged as a key driver not only for establishing individual epithelial cell polarity, but also for coordination of epithelial polarization during apical lumen formation and tissue morphogenesis. In recent years, much work has been dedicated to identifying membrane trafficking regulators required for lumenogenesis. In this review we will summarize the findings from the past couple of decades in defining the molecular machinery governing lumenogenesis both in 3D tissue culture models and during organ development in vivo.

Planar polarized Rab35 functions as an oscillatory ratchet during cell intercalation in the Drosophila epithelium.

The coordination between membrane trafficking and actomyosin networks is essential to the regulation of cell and tissue shape. Here, we examine Rab protein distributions during Drosophila epithelial tissue remodeling and show that Rab35 is dynamically planar polarized. Rab35 compartments are enriched at contractile interfaces of intercalating cells and provide the first evidence of interfacial monopolarity. When Rab35 function is disrupted, apical area oscillations still occur and contractile steps are observed. However, contractions are followed by reversals and interfaces fail to shorten, demonstrating that Rab35 functions as a ratchet ensuring unidirectional movement. Although actomyosin forces have been thought to drive interface contraction, initiation of Rab35 compartments does not require Myosin II function. However, Rab35 compartments do not terminate and continue to grow into large elongated structures following actomyosin disruption. Finally, Rab35 represents a common contractile cell-shaping mechanism, as mesoderm invagination fails in Rab35 compromised embryos and Rab35 localizes to constricting surfaces.Various stages of tissue morphogenesis involve the contraction of epithelial surfaces. Here, the authors identify the Rab GTPase Rab35 as an essential component of this contractile process, which functions as a membrane ratchet to ensure unidirectional movement of intercalating cells.

Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila.

Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression.