Biology subjects

Deretic, J.

Publications and source records attributed to Deretic, J..

6 recordsLinked to original sources

bioRxiv2026

CCDC66 couples actin-microtubule crosslinking to KANK1-associated microtubule targeting and focal adhesion turnover

Cell migration requires actin-based protrusion to be coordinated with microtubule (MT)-dependent focal adhesion (FA) remodeling. Although KANK proteins link talin-containing FAs to cortical MT-capture machinery, how this machinery couples to force-bearing actin networks remains unclear. Here, we identify the ciliopathy-associated protein CCDC66 as an actin-MT crosslinker required for FA turnover and migration. CCDC66 depletion impaired collective, Transwell, and random migration, whereas CCDC66 overexpression enhanced wound closure and Matrigel invasion. Mechanistically, CCDC66 loss reduced MT targeting to peripheral FAs and shifted cells into a ROCK-dependent contractile state marked by impaired lamellipodial protrusion, stress fiber accumulation, enlarged long-lived adhesions, and increased RhoA-ROCK signaling. ROCK or formin inhibition suppressed this state, whereas Rac1 activation failed to restore productive protrusion, indicating that CCDC66 maintains the balance between protrusive and contractile actin organization. In vitro TIRF reconstitution demonstrated that purified CCDC66 directly crosslinks actin filaments and MTs. In cells, CCDC66 associated with KANK1, supported its peri-adhesion organization, and functioned with KANK1 in an overlapping migration pathway. TCGA analyses further revealed that a coordinated CCDC66/KANK/ROCK-associated module, but not CCDC66 expression alone, stratified outcome in chromophobe renal cell carcinoma. These findings reveal a non-ciliary role for CCDC66 in coupling actin-MT integration to FA turnover and contractile-state control, with relevance to developmental disease and cancer-associated motility.

cell biology↗

bioRxiv2026

The Joubert syndrome protein CSPP1 is a conserved regulator of vertebrate multiciliogenesis and motile cilia function

Cilia are conserved microtubule-based organelles required for signaling and fluid transport, and their dysfunction causes ciliopathies. Clinical overlap between sensory and motile ciliopathies suggests that primary and motile ciliogenesis depend on shared regulatory modules. Here, we identify Centrosome and Spindle Pole-associated Protein 1 (CSPP1), a microtubule-associated protein mutated in the neurodevelopmental ciliopathy Joubert syndrome, as a conserved regulator of vertebrate multiciliogenesis. Using mouse tracheal epithelial cultures and Xenopus embryonic epidermis, we show that CSPP1 localizes to fibrous granules and deuterosomes during centriole amplification, and to basal bodies and ciliary axonemes in differentiated multiciliated cells. Loss of CSPP1 impairs centriole amplification, basal body apical migration, spacing, and rotational polarity, and is accompanied by disorganization of the apical microtubule network. CSPP1 depletion also disrupts axoneme assembly, resulting in fewer and shorter cilia with ultrastructural defects, reduced ciliary beating, and impaired cilia driven fluid flow in vivo. Together, our findings identify CSPP1 as a conserved regulator of multiciliogenesis and motile cilia function and establish a basis for future work on how shared cytoskeletal pathways may underlie overlapping features of sensory and motile ciliopathies.

cell biology↗

bioRxiv2024

Ccdc66 regulates primary cilium stability, disassembly and signaling important for epithelial organization

The primary cilium is a conserved, microtubule-based organelle that transduces signaling pathways essential for development and homeostasis. It is a dynamic structure that assembles and disassembles in response to intrinsic and extrinsic stimuli while maintaining remarkable stability and tightly controlled length. Although cilium assembly is well-understood, less is known about the molecular players and pathways governing their stability, length and disassembly. Here, we elucidated the function of Ccdc66, a microtubule-associated protein linked to ciliopathies, in cilium maintenance and disassembly in mouse epithelial cells. We found that Ccdc66 depletion disrupts cilium disassembly, length and stability, but does not affect assembly in these cells. Live imaging of these processes revealed that cilia in Ccdc66-depleted cells frequently fluctuate in length and exhibit increased ectocytosis from the cilium tip. Phenotypic rescue experiments and in vitro microtubule stabilization assays showed that Ccdc66 mediates these functions via regulating the stability of microtubules. Temporal proximity mapping of CCDC66 identified potential new regulators and molecular pathways involved in cilium disassembly. Additionally, depletion of CCDC66 compromised Hedgehog and Wnt pathway activation and disrupted epithelial cell organization and polarity in two-dimensional and three-dimensional cultures. Collectively, our results define Ccdc66 as a new microtubule-stabilizing factor that regulates cilium stability and disassembly, providing insights into the mechanisms of cilium homeostasis and the pathologies associated with Ccdc66.

cell biology↗

bioRxiv2022

The ciliopathy protein CCDC66 controls mitotic progression and cytokinesis by promoting microtubule nucleation and organization

Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and non-dividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the spindle poles, the bipolar spindle, the spindle midzone, the central spindle and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and positioning, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the spindle poles and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provides new insight into nonciliary defects that underlie ciliopathies.

cell biology↗

bioRxiv2022

CCDC66 regulates primary cilium length and signaling competence via multi-site interactions with transition zone and axonemal proteins

The primary cilium is a conserved microtubule-based organelle that serves as a hub for many signaling pathways. It functions as part of the centrosome/cilium complex, which also contains the basal body and the centriolar satellites. Little is known about the mechanisms by which the microtubule-based axoneme of the cilium is assembled with proper length and structure, particularly in terms of the activity of microtubule-associated proteins (MAPs) and the crosstalk between the different compartments of the centrosome/cilium complex. Here, we analyzed CCDC66, a MAP implicated in cilium biogenesis and ciliopathies affecting eye and brain. Live-cell imaging revealed that CCDC66 compartmentalizes between centrosomes, centriolar satellites, and the ciliary axoneme and tip during cilium assembly and disassembly. CCDC66 loss-of-function in human cells causes defects in cilium assembly, length and morphology. Notably, CCDC66 interacts with the MAPs and ciliopathy proteins CEP104 and CSPP1 and cooperates with them during axonemal length regulation. Moreover, CCDC66 interacts with the transition zone protein CEP290 selectively at the centriolar satellites. Its loss disrupts basal body recruitment of transition zone proteins and IFT-B machinery and causes defective Hedgehog signaling. Overall, our results establish CCDC66 as a multifaceted regulator of the primary cilium, and propose a mechanistic insight into how the cooperation of ciliary MAPs as well as subcompartments ensures assembly of a functional cilium.

cell biology↗

bioRxiv2021

ENKD1 is a centrosomal and ciliary microtubule-associated protein important for primary cilium assembly and Hedgehog signaling

Centrioles and cilia are conserved, microtubule-based structures critical for cell function and development. Their structural and functional defects cause cancer and developmental disorders. How microtubules are organized into ordered structures by microtubule-associated proteins (MAPs) and tubulin modifications is best understood during mitosis but is largely unexplored for the centrioles and the ciliary axoneme, which are composed of remarkably stable microtubules that maintain their length at steady state. In particular, we know little about the identity of the centriolar and ciliary MAPs and how they work together during the assembly and maintenance of the cilium and centriole. Here, we identified Enkurin domain containing 1 (ENKD1) as a component of the centriole wall and the axoneme in mammalian cells, and showed that it has extensive proximity interactions with these compartments and MAPs. Using in vitro and cellular assays, we found that ENKD1 is a new MAP that promotes microtubule polymerization and regulates microtubule organization and stability. Consistently, overexpression of ENKD1 increased tubulin polymerization and acetylation and disrupted microtubule organization. Cells depleted for ENKD1 were defective in ciliary length and content regulation and failed to respond to Hedgehog pathway activation. Together, our results establish ENKD1 as a new centriolar and ciliary MAP that regulate primary cilium structure and function, and advances our understanding of the functional and regulatory relationship between MAPs and the primary cilium.

cell biology↗