Biology subjects

Huranova, M.

Publications and source records attributed to Huranova, M..

9 recordsLinked to original sources

bioRxiv2026

Bardet-Biedl Syndrome 1 Mutations Differentially Impact BBSome Integrity and its Function in Ciliary Trafficking

Bardet-Biedl Syndrome (BBS) is a pleiotropic ciliopathy marked by retinal degeneration, obesity, polydactyly, renal and reproductive anomalies, and cognitive impairment. BBS1, the most frequently mutated gene in BBS, encodes a key component of the BBSome complex, which is essential for ciliary membrane trafficking. Although BBS1 is known to be essential for proper BBSome function, the effects of disease-associated BBS1 variants on its activity remain incompletely understood. In this study, we examined how patient-derived BBS1 mutations affect BBSome integrity and its role in cargo transport within primary cilia. Our results show that particular BBS1 mutations interfere with distinct stages of BBSome assembly and trafficking. While M390R disrupts initial pre-BBSome assembly at pericentriolar satellites, E224K impairs both the maturation of the pre-BBSome into the BBSome and its movement from pericentriolar satellites to the cilium. In contrast, the R160Q variant preserves BBSome assembly and permits its localization to cilia. It specifically weakens the BBSome-GPCR interaction mediated by TOM1L2, resulting in defective GPR161 export and increased ciliary IFT turnover. Overall, our study establishes a mechanistic framework linking specific BBS1 mutations to distinct defects in BBSome assembly and function. This framework defines functional classes of BBS1 variants and provides deeper insight into the molecular mechanism and severity of Bardet-Biedl Syndrome.

cell biology↗

bioRxiv2025

Kinesin-3 KIF14 Regulates Intraflagellar Transport Dynamics in Primary Cilia

The primary cilium is an antenna-like organelle that plays a crucial role in development and homeostasis. Its growth and functions depend on the transport of cargo from the cilia base to the tip by intraflagellar transport (IFT) proteins and kinesin-2 motors. Primary cilia exhibit great variation in morphology and function across cell types and organisms. This diversity is thought to be conferred by the modulation of IFT by additional factors. However, examples of such non-canonical regulators, such as kinesin motors distinct from kinesin-2, are sparse. Thus, the involvement of non-canonical ciliary kinesins in intraciliary transport is unclear. Here, we show that a poorly characterized member of the kinesin-3 family, KIF14, plays a prominent role in primary cilia trafficking in human cells. Using live cell imaging and expansion microscopy, we demonstrate that KIF14 depletion leads to impaired IFT. Furthermore, using TIRF microscopy we show that the motility of KIF14 and its effects on cilia trafficking rely on the motor domain of KIF14, which drives the processive runs along the ciliary axoneme in cells and in vitro, in co-operation with the C-terminal CC1 domain. Finally, we demonstrate that C-terminal truncations of KIF14, including patient mutation Q1380x, disrupt IFT by causing traffic jam-like accumulations of ciliary components in the distal part of the primary cilia, leading to bulged tips of cilia. In summary, our data exemplify the role of a non-canonical ciliary kinesin in the regulation of ciliary trafficking and suggest a new paradigm for how kinesin-related trafficking defects may contribute to the pathology of disorder with a ciliary contribution.

cell biology↗

bioRxiv2023

De-suppression of mesenchymal cell identities and variable phenotypic outcomes associated with knockout of Bbs1.

Bardet-Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue specific, and gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane trafficking and intraflagellar transport (IFT) adaptor protein complex formed by 8 BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically-defined clones. Our results suggest that BBS1 is required for suppression of mesenchymal cell identities as IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, and BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively this work suggests that the dynamic stability of the BBSome is essential for suppression of mesenchymal cell identities as epithelial cells differentiate.

genetics↗

bioRxiv2023

BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of the BBSome, a cargo adaptor essential for export of transmembrane receptors from cilia. Although actin-dependent ectocytosis has been proposed as compensatory cellular process for defective cargo retrieval, the underlying molecular mechanism is poorly understood, particularly in the context of BBS pathology. In this study, we investigated how actin polymerization and ectocytosis are regulated within the cilium. Our findings reveal that ciliary CDC42, a RHO-family GTPase triggers in situ actin polymerization, ciliary ectocytosis, and cilia shortening in BBSome-deficient cells. Furthermore, activation of the Sonic Hedgehog pathway further enhances CDC42 activity in BBSome-deficient cilia, but not in healthy cells. Inhibition of CDC42 decreased the frequency and duration of ciliary actin polymerization events and lead to the accumulation of the GPR161 receptor in bulges along the axoneme in BBSome-deficient cells during Sonic Hedgehog signaling. Overall, our study identifies CDC42 as a key trigger of ciliary ectocytosis. Moreover, the hyperactivated ciliary CDC42-actin-ectocytosis axis in BBSome-deficient cells results in cilia shortening and loss of ciliary material, potentially impacting disease severity.

cell biology↗

bioRxiv2020

The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells

Bardet-Biedl Syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. Most BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, BBSome, which mediates the trafficking of ciliary cargoes. Although, the structure of the BBSome has been resolved recently, the mechanism of assembly of this complicated complex in living cells is poorly understood. We generated a large library of human retinal epithelial cell lines deficient in particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We performed a comprehensive analysis of these cell lines using biochemical and microscopy approaches. Our data revealed that the BBSome formation is a sequential process including a step of the pre-BBSome assembly at pericentriolar satellites nucleated by BBS4, followed by the translocation of the BBSome into the ciliary base mediated by BBS1.

cell biology↗

bioRxiv2020

Prospects and limitations of expansion microscopy in chromatin ultrastructure determination

Expansion Microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices. Expansion of plant-derived samples is still at the beginning, and little is known whether the chromatin ultrastructure becomes altered by physical expansion. In this study, we expanded isolated barley nuclei and compared whether ExM can provide a structural view of chromatin comparable with super-resolution microscopy. Different fixation and denaturation/digestion conditions were tested to maintain the chromatin ultrastructure. We achieved up to [~]4.2-times physically expanded nuclei corresponding to a maximal resolution of [~]50-60 nm when imaged by wild-field (WF) microscopy. By applying structured illumination microscopy (SIM, super-resolution) doubling the WF resolution the chromatin structures were observed at a resolution of [~]25-35 nm. WF microscopy showed a preserved nucleus shape and nucleoli. Moreover, we were able to detect chromatin domains, invisible in unexpanded nuclei. However, by applying SIM we observed that the preservation of the chromatin ultrastructure after expansion was not complete and that the majority of the tested conditions failed to keep the ultrastructure. Nevertheless, using expanded nuclei we detected successfully centromere repeats by fluorescence in situ hybridization (FISH) and the centromere-specific histone H3 variant CENH3 by indirect immunostaining. However, although these repeats and proteins were localized at the correct position within the nuclei (indicating a Rabl orientation) their ultrastructural arrangement was impaired.

cell biology↗

bioRxiv2020

Altered hematopoietic system and self-tolerance in Bardet-Biedl Syndrome

Bardet-Biedl Syndrome (BBS) is a pleiotropic genetic disease caused by dysfunction of primary cilia. The immune system of patients with BBS or another ciliopathy has not been investigated, most likely because hematopoietic cells do not form cilia. However, there are multiple indications that the impairment of the processes typically associated with cilia might influence the hematopoietic compartment and immunity. In this study, we analyzed clinical data of BBS patients as well as a corresponding mouse model of BBS4 deficiency. We uncovered that BBS patients have higher incidence of certain autoimmune diseases. BBS patients and animal models have elevated white blood cell levels and altered red blood cell and platelet compartments. Moreover, we observed that BBS4 deficiency alters the development and homeostasis of B cells in mice. Some of the hematopoietic system alterations were caused by the BBS-induced obesity. Overall, our study reveals a connection between a ciliopathy and the alterations of the immune system and the hematopoietic compartment.

genetics↗

bioRxiv2019

Cep97 Is Required For Centriole Structural Integrity And Cilia Formation In Drosophila

Centrioles are highly elaborate microtubule-based structures responsible for the formation of centrosomes and cilia. Despite considerable variation across species and tissues, within any given tissue their size is essentially constant [1, 2]. While the diameter of the centriole cylinder is set by the dimensions of the inner scaffolding structure of the cartwheel [3], how centriole length is set so precisely and stably maintained over many cell divisions is not well understood. Cep97 and CP110 are conserved proteins that localize to the distal end of centrioles and have been reported to limit centriole elongation in vertebrates [4, 5]. Here, we examine Cep97 function in Drosophila melanogaster. We show that Cep97 is essential for formation of full-length centrioles in multiple tissues of the fly. We further identify the microtubule deacetylase Sirt2 as a Cep97 proximity interactor. Deletion of Sirt2 likewise affects centriole size. Interestingly, so does deletion of the acetylase Atat1, indicating that loss of stabilizing acetyl marks impairs centriole integrity. Cep97 and CP110 were originally identified as inhibitors of cilia formation in vertebrate cultured cells [6] and loss of CP110 is a widely used marker of basal body maturation. In contrast, in Drosophila Cep97 is only transiently removed from basal bodies and loss of Cep97 strongly impairs ciliogenesis. Collectively, our results support a model whereby Cep97 functions as part of a protective cap that acts together with the microtubule acetylation machinery to maintain centriole stability, essential for proper function in cilium biogenesis.

cell biology↗

bioRxiv2017

Strong homeostatic TCR signals induce formation of self-tolerant virtual memory CD8 T cells

Virtual memory T cells are foreign antigen-inexperienced T cells that have acquired memory-like phenotype and constitute for 10-20% of all peripheral CD8+ T cells in mice. Their origin, biological roles, and relationship to naive and foreign antigen-experienced memory T cells are incompletely understood. By analyzing TCR repertoires and using retrogenic monoclonal T-cell populations, we show that virtual memory T cells originate exclusively from strongly self-reactive T cells. Moreover, we show that the stoichiometry of the CD8 interaction with Lck regulates the size of the virtual memory T-cell compartment via modulating the self-reactivity of individual T-cell clones. We propose a so far unappreciated peripheral T-cell fate decision checkpoint that eventually leads to the differentiation of highly self-reactive T cells into virtual memory T cells. This underlines the importance of the variable level of self-reactivity in polyclonal T cells for the generation of functional T-cell diversity. Although virtual memory T cells descend from the highly self-reactive clones and acquire a partial memory program, they do not show higher capacity to induce autoimmune diabetes than naive T cells. Thus, virtual memory T cells are not generally more responsive than naive T cells, because their activity highly depends on the immunological context.\n\nSummaryWe conclude that virtual memory T cells are formed from self-reactive CD8+ T cells in a process regulated by CD8-Lck stoichiometry. Despite their self-reactivity and partial memory differentiation program, virtual memory T cells did not show a strong autoimmune potential.

immunology↗