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Microtubules in renal tubule epithelial cells. (a) Immunostaining for α-tubulin, Megalin, and nuclei in WT and Camsap3dc/dc kidneys at P21. (b) Quantification of α-tubulin intensity in PCT and DCT/CD of WT kidneys (PCT, n = 28 tubules; DCT/CD, n = 18 tubules) and Camsap3dc/dc kidneys (PCT, n = 13 tubules; DCT/CD, n = 9 tubules). Error bars indicate S.D. ***p < 0.001, t-test. (c) Immunostaining for α-tubulin, CAMSAP3, and DAPI in WT and Camsap3dc/dc kidneys at P21. Arrowheads indicate CAMSAP3-dc proteins anchoring to unidentified structures. The apical localization of CAMSAP3 was absent in Camsap3dc/dc cells. See also Figure S2D.
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Epithelial cells organize an ordered array of non-centrosomal microtubules, the minus ends of which are regulated by CAMSAP3. The role of these microtubules in epithelial functions, however, is poorly understood. Here, we show that the kidneys of mice in which Camsap3 is mutated develop cysts at the proximal convoluted tubules (PCTs). PCTs were sev...
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... In fact, mutant Camsap3 dc/dc transgenic mice expressing a truncated CAMSAP3 protein with a deleted C-terminal microtubule-binding domain in proximal renal tubules developed significant cystic disease. (88) This member of the calmodulin-regulated spectrin-associated protein (CAMSAP)/Nezha/Patronin family is essential for epithelial microtubule organization. In Camsap3 mutant mice, nuclear PIEZO1 was significantly increased both in cells from was not certified by peer review) is the author/funder. ...
The disruption of calcium signaling associated with polycystin deficiency has been proposed as the primary event underlying the increased abnormally patterned epithelial cell growth characteristic of Polycystic Kidney Disease. Calcium can be regulated through mechanotransduction, and the mechanosensitive cation channel Piezo1 has been implicated in sensing of intrarenal pressure and in urinary osmoregulation. However, a possible role for PIEZO1 in kidney cystogenesis remains undefined. We hypothesized that cystogenesis in ADPKD reflects altered mechanotransduction, suggesting activation of mechanosensitive cation channels as a therapeutic strategy for ADPKD. Here, we show that Yoda-1 activation of PIEZO1 increases intracellular Ca ²⁺ and reduces forskolin-induced cAMP levels in mIMCD3 cells. Yoda-1 reduced forskolin-induced IMCD cyst surface area in vitro and in mouse metanephros ex vivo in a dose-dependent manner. Knockout of polycystin-2 dampened the efficacy of PIEZO1 activation in reducing both cAMP levels and cyst surface area in IMCD3 cells. However, collecting duct-specific Piezo1 knockout neither induced cystogenesis in wild-type mice nor affected cystogenesis in the Pkd1 RC/RC model of ADPKD. Our study suggests that polycystin-2 and PIEZO1 play a role in mechanotransduction during cystogenesis in vitro , and ex vivo , but that in vivo cyst expansion may require inactivation or repression of additional suppressors of cystogenesis and/or growth. Our study provides a preliminary proof of concept for PIEZO1 activation as a possible component of combination chemotherapy to retard or halt cystogenesis and/or cyst growth.
... Ранее уже указывалось на роль микротрубочек в патогенезе развития патологии канальцевого аппарата. Необходимо отметить, что CAMSAP3 (Calmodulin-regulated spectrin-associated protein 3) принимает участие в правильной ориентации микротрубочек внутри эпителиальной клетки канальцев [36]. CAMSAP3 относится к семейству белков, регулируемый кальмодулином и спектрином, главной функцией которого является связывание отрицательно заряженных полюсов эпителиальной клетки канальца. ...
Currently, there is a high scientific interest in studying the features of the structure and functions of the tubules of the kidneys. The relevance of the topic is due to the potential possibility of identifying various markers of tubular dysfunction and using them for early diagnosis of not only tubulopathies, but also glomerular disorders. In clinical practice, markers of tubular dysfunction are used insufficiently. The article presents information about the anatomical and functional features of the proximal and distal parts of the tubular apparatus, outlines highly organized mechanisms of intermolecular interaction, presents the main biologically active substances, the change in the concentration of which is a consequence of damage to the tubules. The presented manuscript is the product of a deep analysis and systematization of the available data in Russian and foreign information and analytical portals.
... Therefore, both kidneys were prepared out of euthanized WT and cKO mice with ages of P22-P339. Since the kidney-to-body weight ratio has been commonly used to predict kidney function (Hughes et al., 1999;Mitsuhata et al., 2021), we weighed 19 WT mice and 7 Camsap3-cKO mice and their kidneys. Kidney weight as a proportion of body weight was recorded. ...
... Exons 14-17 encode the key MT binding domain of CAMSAP3, CKK domain. Because the mutant CAMSAP3 without the CKK domain is incapable of binding to MTs to form proper MT network in the epithelial cells in kidney, Camsap3 dc/dc mice developed malfunction of kidneys with cyst at the proximal convoluted tubules starting at E17.5 even though kidney had normal appearance at P21 (Mitsuhata et al., 2021). Since renal dysfunction often caused body weight loss (Hickman and Swan, 2010), the deterioration of kidneys observed in Camsap3-cKO may contribute to the small body size of Camsap3-cKO mice older than 8-month (Figure 4). ...
Kinocilia are exceptionally long primary sensory cilia located on vestibular hair cells, which are essential for transmitting key signals that contribute to mammalian balance and overall vestibular system function. Kinocilia have a “9+2” microtubule (MT) configuration with nine doublet MTs surrounding two central singlet MTs. This is uncommon as most mammalian primary sensory cilia have a “9+0” configuration, in which the central MT pair is absent. It has yet to be determined what the function of the central MT pair is in kinocilia. Calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) regulates the minus end of MTs and is essential for forming the central MT pair in motile cilia, which have the “9+2” configuration. To explore the role of the central MT pair in kinocilia, we created a conditional knockout model (cKO), Camsap3-cKO, which intended to eliminate CAMSAP3 in limited organs including the inner ear, olfactory bulb, and kidneys. Immunofluorescent staining of vestibular organs demonstrated that CAMSAP3 proteins were significantly reduced in Camsap3-cKO mice and that aged Camsap3-cKO mice had significantly shorter kinocilia than their wildtype littermates. Transmission electron microscopy showed that aged Camsap3-cKO mice were in fact missing that the central MT pair in kinocilia more often than their wildtype counterparts. In the examination of behavior, wildtype and Camsap3-cKO mice performed equally well on a swim assessment, right-reflex test, and evaluation of balance on a rotarod. However, Camsap3-cKO mice showed slightly altered gaits including reduced maximal rate of change of paw area and a smaller paw area in contact with the surface. Although Camsap3-cKO mice had no differences in olfaction from their wildtype counterparts, Camsap3-cKO mice did have kidney dysfunction that deteriorated their health. Thus, CAMSAP3 is important for establishing and/or maintaining the normal structure of kinocilia and kidney function but is not essential for normal olfaction. Our data supports our hypothesis that CAMSAP3 is critical for construction of the central MT pair in kinocilia, and that the central MT pair may be important for building long and stable axonemes in these kinocilia. Whether shorter kinocilia might lead to abnormal vestibular function and altered gaits in older Camsap3-cKO mice requires further investigation.
... 1995), to the membrane, and downregulation of CAMSAP3 reduces the capacity of the embryo to form pluripotent cells . Whether this reduction is co-triggered by the ability of CAMSAP3 to regulate Yes-associated protein (YAP) activity (Mitsuhata et al., 2021), which is a crucial transcription factor required for lineage segregation in the mammalian embryo, remains to be determined. Given the large number of CAMSAP3 interactors ( Fig. 1), its full potential in regulating early mammalian embryogenesis may not yet be recognised. ...
... Despite these seminal findings, there is a profound need to advance our understanding regarding the spatial real-time organisation of organelles regulating mammalian embryogenesis. Known to coordinate endosome trafficking (Khanal et al., 2016;Wong et al., 2018;Zenker et al., 2017), Golgi construction and positioning (Toya et al., 2016;Wang et al., 2017), as well as mitochondrial shape (Mitsuhata et al., 2021), CAMSAP3 has the ability to emerge as a master regulator for such processes. ...
With the advancement of cutting-edge live imaging technologies, microtubule remodelling has evolved as an integral regulator for the establishment of distinct differentiated cells. However, despite their fundamental role in cell structure and function, microtubules have received less attention when unravelling the regulatory circuitry of pluripotency. Here, we summarise the role of microtubule organisation and microtubule-dependent events required for the formation of pluripotent cells in vivo by deciphering the process of early embryogenesis: from fertilisation to blastocyst. Furthermore, we highlight current advances in elucidating the significance of specific microtubule arrays in in vitro culture systems of pluripotent stem cells and how the microtubule cytoskeleton serves as a highway for the precise intracellular movement of organelles. This Review provides an informed understanding of the intrinsic role of subcellular architecture of pluripotent cells and accentuates their regenerative potential in combination with innovative light-inducible microtubule techniques.
... CAMSAP (calmodulin-regulated spectrin-associated protein) and its invertebrate homologs form a protein family, each member of which binds the minus end of non-centrosomal microtubules (Meng et al., 2008;Baines et al., 2009;Goodwin and Vale, 2010;Tanaka et al., 2012;Hendershott and Vale, 2014;Jiang et al., 2014;King et al., 2014;Richardson et al., 2014). Through this property of CAMSAP, it regulates the dynamics of the minus ends of microtubules, simultaneously anchoring them to particular subcellular sites (Nashchekin et al., 2016;Toya et al., 2016), and contributes to a variety of cell and tissue morphogenesis (Noordstra et al., 2016;Martin et al., 2018;Takeda et al., 2018;Ko et al., 2019;Kimura et al., 2021;Mitsuhata et al., 2021), including neuritegenesis (Chuang et al., 2014;Yau et al., 2014;Pongrakhananon et al., 2018;Feng et al., 2019;Wang et al., 2019;Chen et al., 2020). ...
... This characteristic microtubule assembly is disrupted in mice bearing a mutant gene encoding a truncated Camsap3 (Camsap3 dc/dc mice), in which the CKK domain is deleted, because mRNA transcribed from the mutated gene does not cover the exons that encode the CKK domain (Toya et al., 2016). Camsap3 dc/dc mice show other defects, such as cyst formation in proximal renal tubules (Mitsuhata et al., 2021), and narrowing of the lateral ventricles in the brain (Kimura et al., 2021), although the overall brain architecture looks normal. Camsap3 dc/dc mice and Camsap3 null mutant mice show similar abnormalities (Pongrakhananon et al., 2018;Mitsuhata et al., 2021), suggesting that CAMSAP3 lacking a CKK domain is fully non-functional. ...
... Camsap3 dc/dc mice show other defects, such as cyst formation in proximal renal tubules (Mitsuhata et al., 2021), and narrowing of the lateral ventricles in the brain (Kimura et al., 2021), although the overall brain architecture looks normal. Camsap3 dc/dc mice and Camsap3 null mutant mice show similar abnormalities (Pongrakhananon et al., 2018;Mitsuhata et al., 2021), suggesting that CAMSAP3 lacking a CKK domain is fully non-functional. ...
Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body, which are linked by a ‘transition zone’. The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that CAMSAP3, a protein that can stabilize the minus end of a microtubule, concentrates at multiple sites of the cilium–basal body complex, including the upper region of the transition zone or the axonemal basal plate where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the basal plate, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme, and thereby supports the coordinated motion of multicilia in airway epithelial cells.
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... /2021 CAMSAP (calmodulin-regulated spectrin-associated protein) and its invertebrate relatives form a protein family, each member of which binds the minus end of noncentrosomal microtubules (Baines et al., 2009;Goodwin and Vale, 2010;Hendershott and Vale, 2014;Jiang et al., 2014;King et al., 2014;Meng et al., 2008;Richardson et al., 2014;Tanaka et al., 2012). Through this property of CAMSAP, it regulates the dynamics of the minus ends of microtubules, simultaneously anchoring them to particular subcellular sites (Nashchekin et al., 2016;Toya et al., 2016), and contributes to a variety of cell and tissue morphogenesis (Kimura et al., 2021;Ko et al., 2019;Martin et al., 2018;Mitsuhata et al., 2021;Noordstra et al., 2016;Takeda et al., 2018), including neuritegenesis (Chen et al., 2020;Chuang et al., 2014;Feng et al., 2019;Pongrakhananon et al., 2018;Wang et al., 2019;Yau et al., 2014). In vertebrate epithelial cells, CAMSAP3, a member of this molecular family, is concentrated at apical cortical regions, tethering non-centrosomal microtubules to these sites (Toya et al., 2016), resulting in the assembly of microtubules whose plus ends point basally. ...
... ; https://doi.org/10.1101/2021.04.21.440849 doi: bioRxiv preprint microtubules for displaying a clustered appearance in certain epithelial cells (Tanaka et al., 2012;Toya et al., 2016). On the other hand, the CKK-deficient CAMSAP3 puncta were still detectable at the #3 site (and also partly at the #2 site) of airway cells, as observed in some other tissues (Kimura et al., 2021;Mitsuhata et al., 2021), implying the possibility that CAMSAP3 is able to cluster independently of microtubules, when it binds particular subcellular structures or molecules. ...
Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body, which are linked by a ‘transition zone’. The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here, using superresolution microscopy, we show that CAMSAP3, a protein that can stabilize the minus end of a microtubule, concentrates at multiple sites of the cilium-basal body complex, including the upper region of the transition zone or the axonemal basal plate where the central pair of microtubules (CP) terminates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the basal plate, as well as the failure of multicilia to undergo synchronized beating. These findings indicate that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP, and thereby supports the coordinated motion of multicilia in airway epithelial cells.
Piezo1 is an essential mechanosensitive transduction ion channel in mammals. Its unique structure makes it capable of converting mechanical cues into electrical and biological signals, modulating biological and (patho)physiological processes in a wide variety of cells. There is increasing evidence demonstrating that the piezo1 channel plays a vital role in renal physiology and disease conditions. This review summarizes the current evidence on the structure and properties of Piezo1, gating modulation, and pharmacological characteristics, with special focus on the distribution and (patho)physiological significance of Piezo1 in the kidney, which may provide insights into potential treatment targets for renal diseases involving this ion channel.
Renal cysts can be both an independent nosological entity, and a clinical manifestation, or a complication of a severe diseases. Understanding the epidemiology, pathogenesis and diversity of causes of cystic kidney disease contributes to the timely diagnosis and selection of reasonable treatment and prevention tactics. The presented literature review describes the most common processes that contribute to the development of this pathology, as well as genetic diseases and rare causes, the clinical manifestation of which is the formation of cystic cavities. The article presents the main diagnostic algorithms and modern classifications of the disease for practical assistance to the doctor. The review contains up-to-date information on the modern staging of cystic kidney disease according to the Bosniak classification, and also presents the risk of malignancy, according to the statistical data presented in the literature.
Technological advancement to enhance tumor cells (TC) has allowed discovery of various cellular bio-markers: cancer stem cells (CSC), circulating tumor cells (CTC), and endothelial progenitor cells (EPC). These are responsible for resistance, metastasis, and premetastatic conditions of cancer. Detection of CSC, CTC, and EPC assists in early diagnosis, recurrence prediction, and treatment efficacy. This review describes various methods to detect TC subpopulations such as in vivo assays (sphere-forming, serial dilution, and serial transplantation), in vitro assays (colony-forming cells, microsphere, side-population, surface antigen staining, aldehyde dehydrogenase activity, and Paul Karl Horan label-retaining cells, surface markers, nonenriched and enriched detection), reporter systems, and other analytical methods (flow cytometry, fluorescence microscopy/spectroscopy, etc.). The detailed information on methods to detect CSC, CTC, and EPC in this review will assist investigators in successful prognosis, diagnosis, and cancer treatment with greater ease.
