Biology subjects

Jewett, C. E.

Publications and source records attributed to Jewett, C. E..

10 recordsLinked to original sources

bioRxiv2026

Trisomy 21 alters ciliary localization of Sonic Hedgehog signaling proteins

Trisomy 21 (T21), the cause of Down syndrome, impairs cilia-dependent Sonic Hedgehog (SHH) signaling. We investigated how T21 affects SHH signaling at the primary cilium and found that Smoothened (SMO) protein - the main effector of SHH signaling - is diminished in T21 primary cilia. Specifically, T21 cells exhibit both decreased SMO localization to cilia and reduced ciliary SMO phosphorylation (pSMO). Our findings suggest that reduced ciliary SMO in T21 results from defective entry into or maintenance in the cilium rather than impaired transport to centrosomes. Moreover, unlike the ciliogenesis defects observed in T21, the SMO defect does not appear to be associated with increased PCNT protein abundance. We also found that GPR161 (a negative SHH regulator), exhibits increased ciliary localization in T21. Notably, prolonged serum depletion to promote primary cilia maturation equalizes ciliary levels of SMO and GPR161 between T21 cells and D21 controls. We propose that delayed primary cilia maturation contributes to defective SHH signaling in T21. SUMMARYT21 impedes primary cilia assembly and disrupts SHH signaling by diminishing ciliary SMO and increasing GPR161, but extended ciliary maturation equalizes SHH protein localization between D21 and T21.

cell biology↗

bioRxiv2025

The DNA damage response pathway is required for multiciliated cell differentiation

DNA damage can result from external sources or occur during programmed genome rearrangements in processes like immunity or meiosis. To maintain genome integrity, cells activate DNA repair pathways that prevent harmful outcomes such as cancer or immune dysfunction. In this study, we uncover a novel role for DNA damage during the terminal differentiation of multiciliated cells (MCCs). MCCs, which line the airways, reproductive tracts, and brain ventricles, produce hundreds of motile cilia, each anchored by a centriole. Therefore, MCCs must generate hundreds of centrioles during differentiation. Normally, centriole duplication is tightly linked to the S and G2 phases of the cell cycle, raising questions about how MCCs override numerical and temporal restrictions on centriole duplication. We find that differentiating MCCs accumulate extensive double-stranded DNA breaks during centriole amplification, with damage levels correlating with centriole number. DNA damage response (DDR) kinases are essential for supporting centriole biogenesis and ciliogenesis. We also observe that transcriptional activity, required for the expression of centriole and cilia genes, produces RNA-DNA hybrids (R-loops) that co-localize with DNA damage. This suggests that transcription-coupled DNA damage helps initiate a pseudo-cell cycle program, permitting centriole amplification without triggering full S/G2 phase processes. Our findings indicate that MCCs harness DDR signaling as part of their developmental program, revealing a broader principle in which the canonical cell cycle is adaptively rewired during differentiation.

cell biology↗

bioRxiv2024

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.

cell biology↗

bioRxiv2024

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.

cell biology↗

bioRxiv2024

Microtubule-dependent orchestration of centriole amplification in brain multiciliated cells

Multiciliated cell (MCC) differentiation is a calibrated version of the canonical cell cycle. The MCC cell cycle variant sustains amplification of centrioles for the nucleation of dozens of motile cilia, while avoiding cell division. In this study, we show that the MCC cell cycle variant is an accelerated version of the canonical cell cycle, which superposes two cycles of centriole biogenesis, in order to obtain multiple mature centrioles within a single -instead of double- cell cycle iteration. We further show that the precocious maturation of amplified procentrioles is even determinant for their spatial self-organization, disengagement and apical migration for cilia nucleation. Our findings collectively suggest that the decomposition of centriole biogenesis over two cycle iterations in dividing cells may have been adopted to ensure the growth of a solitary primary cilium, and exemplify how minimal deviations of the canonical cell cycle allow MCC progenitors to both amplify, and accelerate, centriole biogenesis for vital motile ciliogenesis.

cell biology↗

bioRxiv2023

Lamin B Receptor Upregulation in Metastatic Melanoma Causes Cholesterol-Mediated Nuclear Envelope Fragility

Metastatic cancer cells migrate through regions of tissue confinement, causing nuclear envelope (NE) rupture and heritable DNA damage. We discovered that cells from multiple cancers have increased NE fragility in confinement and transcriptional upregulation of nuclear genes compared to benign counterparts. A bioinformatic-driven siRNA screen revealed that lamin B receptor (LBR) upregulation correlates with melanoma progression and NE fragility. Increased LBR cholesterol synthase activity causes accumulation of cholesterol in the NE, which is necessary and sufficient for nuclear deformability and NE rupture in cells confined in vitro and is associated with NE rupture in invasive cells migrating out of tumor organoids and tumors in vivo. Thus, LBR upregulation causes excess NE cholesterol, driving nuclear fragility in confined migrating melanoma cells, establishing a direct role for nuclear membrane lipid composition in metastatic cancers.

cell biology↗

bioRxiv2022

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.

cell biology↗

bioRxiv2021

Trisomy 21 induces pericentrosomal crowding disrupting early stages of primary ciliogenesis and mouse cerebellar development

Primary cilia are signaling organelles essential for development and homeostasis. Loss of primary cilia is lethal, and decreased or defective cilia cause multisystemic conditions called ciliopathies. Down syndrome shares clinical overlap with ciliopathies. We previously showed that trisomy 21 diminishes primary cilia formation and function due to elevated Pericentrin, a centrosome protein encoded on chromosome 21. Pericentrin is mislocalized, creating aggregates that disrupt pericentrosomal trafficking and microtubule organization. Here, we examine the cilia-related molecules and pathways disrupted in trisomy 21 and their in vivo phenotypic relevance. Utilizing ciliogenesis time course experiments, we reveal how Pericentrin, microtubule networks, and components of ciliary vesicles are reorganized for ciliogenesis in euploid cells. Early in ciliogenesis, chromosome 21 polyploidy results in elevated Pericentrin and microtubule networks away from the centrosome that ensnare MyosinVA and EHD1, blocking mother centriole uncapping that is essential for ciliogenesis. Ciliated trisomy 21 cells have persistent trafficking defects that reduce transition zone protein localization, which is critical for Sonic hedgehog signaling. Sonic hedgehog signaling is decreased and anticorrelates with Pericentrin levels in trisomy 21 primary mouse embryonic fibroblasts. Finally, we observe decreased ciliation in vivo. A mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers. Our work reveals that elevated Pericentrin in trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding results in signaling defects consistent with the neurological deficits found in individuals with Down syndrome.

cell biology↗

bioRxiv2021

Trisomy 21 increases microtubules and disrupts centriolar satellite localization

Trisomy 21, the cause 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. Here we investigate the mechanisms by which PCNT imbalances disrupt cilia. Using isogenic RPE-1 cells with increased chromosome 21 dosage, we find PCNT protein accumulates around the centrosome as a pericentrosomal cluster of enlarged cytoplasmic puncta that localize along and at MT ends. Cytoplasmic PCNT puncta impact the intracellular MT trafficking network required for primary cilia, as the PCNT puncta sequester cargo peripheral to centrosomes in what we call pericentrosomal crowding. The centriolar satellite proteins, PCM1, CEP131 and CEP290, important for ciliogenesis, accumulate at sites of enlarged PCNT puncta in trisomy 21 cells. Reducing PCNT when chromosome 21 ploidy is elevated is sufficient to decrease PCNT puncta, reestablish a normal density of MTs around the centrosome, restore ciliogenesis to wild type levels and decrease pericentrosomal crowding. 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 microtubule network deleterious to normal centriolar satellite distribution. We propose that chromosome 21 aneuploidy disrupts MT-dependent intracellular trafficking required for primary cilia. TOCMcCurdy et al explore why elevated Pericentrin in trisomy 21 negatively impacts primary cilia formation and signaling. They find that elevated Pericentrin produces more pericentrosomal puncta that associates with and increases microtubules. Changes to Pericentrin and microtubules mislocalizes centriolar satellites in a pericentrosomal crowd.

cell biology↗

bioRxiv2020

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, inclusion bodies, and lysosomal accumulation. Single and double mutant analysis suggest that fip5a and fip5b function in parallel and regulate apical 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 biology↗