Uncovering structural themes across cilia microtubule inner proteins with implications for human cilia function.

Centrosomes and cilia are microtubule-based superstructures vital for cell division, signaling, and motility. The once thought hollow lumen of their microtubule core structures was recently found to hold a rich meshwork of microtubule inner proteins (MIPs). To address the outstanding question of how distinct MIPs evolved to recognize microtubule inner surfaces, we applied computational sequence analyses, structure predictions, and experimental validation to uncover evolutionarily conserved microtubule- and MIP-binding modules named NWE, SNYG, and ELLEn, and PYG and GFG-repeat by their signature motifs. These modules intermix with MT-binding DM10-modules and Mn-repeats in 24 Chlamydomonas and 33 human proteins. The modules molecular characteristics provided keys to identify elusive cross-species homologs, hitherto unknown human MIP candidates, and functional properties for seven protein subfamilies, including the microtubule seam-binding NWE and ELLEn families. Our work defines structural innovations that underpin centriole and axoneme assembly and demonstrates that MIPs co-evolved with centrosomes and cilia.

The Interaction between ADK and SCG10 Regulate the Repair of Nerve Damage.

The cytoskeleton must be remodeled during neurite outgrowth, and Superior Cervical Ganglion 10 (SCG10) plays a critical role in this process by depolymerizing Microtubules (MTs), conferring highly dynamic properties to the MTs. However, the precise mechanism of action of SCG10 in the repair of injured neurons remains largely uncertain. Using transcriptomic identification, we discovered that SCG10 expression was downregulated in neurons after Spinal Cord Injury (SCI). Additionally, through mass spectrometry identification, immunoprecipitation, and pull-down assays, we established that SCG10 could interact with Adenosine Kinase (ADK). Furthermore, we developed an excitotoxicity-induced neural injury model and discovered that ADK suppressed injured neurite re-growth, whereas, through overexpression and small molecule interference experiments, SCG10 enhanced it. Moreover, we discovered ADK to be the upstream of SCG10. More importantly, the application of the ADK inhibitor called 5-Iodotubercidin (5-ITu) was found to significantly enhance the recovery of motor function in mice with SCI. Consequently, our findings suggest that ADK plays a negative regulatory role in the repair of injured neurons. Herein, we propose a molecular interaction model of the SCG10-ADK axis to regulate neuronal recovery.

SPAG6 regulates cell proliferation and apoptosis via TGF-ß/Smad signal pathway in adult B-cell acute lymphoblastic leukemia.

Adult B-cell acute lymphoblastic leukemia (B-ALL) prognosis remains unsatisfactory, and searching for new therapeutic targets is crucial for improving patient prognosis. Sperm-associated antigen 6 (SPAG6), a member of the cancer-testis antigen family, plays an important role in tumors, especially hematologic tumors; however, it is unknown whether SPAG6 plays a role in adult B-ALL. In this study, we demonstrated for the first time that SPAG6 expression was up-regulated in the bone marrow of adult B-ALL patients compared to healthy donors, and expression was significantly reduced in patients who achieved complete remission (CR) after treatment. In addition, patients with high SPAG6 expression were older (≥ 35 years; P = 0.015), had elevated white blood cell counts (WBC > 30 × 109/L; P = 0.021), and a low rate of CR (P = 0.036). We explored the SPAG6 effect on cell function by lentiviral transfection of adult B-ALL cell lines BALL-1 and NALM-6, and discovered that knocking down SPAG6 significantly inhibited cell proliferation and promoted apoptosis. We identified that SPAG6 knockdown might regulate cell proliferation and apoptosis via the transforming growth factor-ß (TGF-ß)/Smad signaling pathway.

The C-terminus of stathmin-like proteins governs the stability of their complexes with tubulin.

Microtubule dynamics is modulated by many cellular factors including stathmin family proteins. Vertebrate stathmins sequester two αß-tubulin heterodimers into a tight complex that cannot be incorporated in microtubules. Stathmins are regulated at the expression level during development and among tissues; they are also regulated by phosphorylation. Here, we study the dissociation kinetics of tubulin:stathmin assemblies in presence of different tubulin-binding proteins and identify a critical role of the C-terminus of the stathmin partner. Destabilizing this C-terminal region may represent an additional regulatory mechanism of the interaction with tubulin of stathmin proteins.

The microtubule-severing protein UNC-45A preferentially binds to curved microtubules and counteracts the microtubule-straightening effects of Taxol.

Uncoordinated protein 45A (UNC-45A) is the only known ATP-independent microtubule (MT)-severing protein. Thus, it severs MTs via a novel mechanism. In vitro and in cells, UNC-45A-mediated MT severing is preceded by the appearance of MT bends. While MTs are stiff biological polymers, in cells, they often curve, and the result of this curving can be breaking off. The contribution of MT-severing proteins on MT lattice curvature is largely undefined. Here, we show that UNC-45A curves MTs. Using in vitro biophysical reconstitution and total internal fluorescence microscopy analysis, we show that UNC-45A is enriched in the areas where MTs are curved versus the areas where MTs are straight. In cells, we show that UNC-45A overexpression increases MT curvature and its depletion has the opposite effect. We also show that this effect occurs is independent of actomyosin contractility. Lastly, we show for the first time that in cells, Paclitaxel straightens MTs, and that UNC-45A can counteracts the MT-straightening effects of the drug.

Upregulated SPAG6 correlates with increased STAT1 and is associated with reduced sensitivity of interferon-α response in BCR::ABL1 negative myeloproliferative neoplasms.

Sperm-associated antigen 6 (SPAG6) has been identified as an oncogene or tumor suppressor in various types of human cancer. However, the role of SPAG6 in BCR::ABL1 negative myeloproliferative neoplasms (MPNs) remains unclear. Herein, we found that SPAG6 was upregulated at the mRNA level in primary MPN cells and MPN-derived leukemia cell lines. The SPAG6 protein was primarily located in the cytoplasm around the nucleus and positively correlated with ß-tubulin expression. In vitro, forced expression of SPAG6 increased cell clone formation and promoted G1 to S cell cycle progression. Downregulation of SPAG6 promoted apoptosis, reduced G1 to S phase transition, and impaired cell proliferation and cytokine release accompanied by downregulated signal transducer and activator of transcription 1 (STAT1) expression. Furthermore, the inhibitory effect of interferon-α (INF-α) on the primary MPN cells with high SPAG6 expression was decreased. Downregulation of SPAG6 enhanced STAT1 induction, thus enhancing the proapoptotic and cell cycle arrest effects of INF-α both in vitro and in vivo. Finally, a decrease in SPAG6 protein expression was noted when the STAT1 signaling was blocked. Chromatin immunoprecipitation assays indicated that STAT1 protein could bind to the SPAG6 promoter, while the dual-luciferase reporter assay indicated that STAT1 could promote the expression of SPAG6. Our results substantiate the relationship between upregulated SPAG6, increased STAT1, and reduced sensitivity to INF-α response in MPN.

Cooption of regulatory modules for tektin paralogs during ciliary band formation in a marine annelid larva.

Tektins are a highly conserved family of coiled-coil domain containing proteins known to play a role in structure, stability and function of cilia and flagella. Tektin proteins are thought to form filaments which run the length of the axoneme along the inner surface of the A tubule of each microtubule doublet. Phylogenetic analyses suggest that the tektin family arose via duplications from a single tektin gene in a unicellular organism giving rise to four and five tektin genes in bilaterians and in spiralians, respectively. Although tektins are found in most metazoans, little is known about their expression and function outside of a handful of model species. Here we present the first comprehensive study of tektin family gene expression in any animal system, in the spiralian annelid Platynereis dumerilii. This indirect developing species retains a full ancient spiralian complement of five tektin genes. We show that all five tektins are expressed almost exclusively in known ciliary structures following the expression of the motile cilia master regulator foxJ1. The three older bilaterian tektin-1, tektin-2, and tektin-4 genes, show a high degree of spatial and temporal co-regulation, while the spiralian specific tektin-3/5A and tektin-3/5B show a delay in onset of expression in every ciliary structure. In addition, tektin-3/5B transcripts show a restricted subcellular localization to the most apical region near the multiciliary arrays. The exact recapitulation of the sequence of expression and localization of the five tektins at different times during larval development indicates the cooption of a fixed regulatory and cellular program during the formation of each ciliary band and multiciliated cell type in this spiralian.

Mechanisms of damage to sperm structure in mice on the zinc-deficient diet.

BACKGROUND: Zinc (Zn)is an essential trace element for spermatogenesis and its deficiency causes abnormal spermatogenesis. OBJECTIVE: The present study was conducted to examine the mechanisms by which Zn-deficient diet impairs sperm morphology and its reversibility. METHODS: 30 SPF grade male Kunming (KM) mice were randomly divided into three groups, 10 mice per group. Zn-normal diet group (ZN group) was given Zn-normal diet（Zn content= 30 mg/kg）for 8 weeks. Zn-deficienct diet group (ZD group) was given Zn-deficienct diet（Zn content< 1 mg/kg）for 8 weeks. Zn-deficient and Zn-normal diet group（ZDN group）was given 4 weeks Zn-deficienct diet followed by 4 weeks Zn-normal diet. After 8 weeks, the overnight fasted mice were sacrificed, and blood and organs were collected for further analysis. RESULTS: The experimental results showed that Zn-deficienct diet leads to increased abnormal morphology sperm and testicular oxidative stress.The rate of abnormal morphology sperm, chromomycin A3(CMA3), DNA fragmentation index (DFI), malondialdehyde (MDA) were significantly increased, and a-kinase anchor protein 4(AKAP4), dynein axonemal heavy chain 1(DNAH1), sperm associated antigen 6(SPAG6), cilia and flagella associated protein 44(CFAP44), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), nuclear factor erythroid 2-related factor (NRF2), NAD(P)H:quinone oxidoreductase 1(NQO1)and heme oxygenase 1(HO1) were significantly decreased in the ZD group mice. While the changes in above indicators caused by Zn-deficient diet were significantly alleviated in the ZDN group. CONCLUSION: It was concluded that Zn-deficient diet causes abnormal morphology sperm and testicular oxidative stress in male mice. Abnormal morphology sperm caused by Zn-deficient diet are reversible, and Zn-normal diet can alleviate them.

Native doublet microtubules from Tetrahymena thermophila reveal the importance of outer junction proteins.

Cilia are ubiquitous eukaryotic organelles responsible for cellular motility and sensory functions. The ciliary axoneme is a microtubule-based cytoskeleton consisting of two central singlets and nine outer doublet microtubules. Cryo-electron microscopy-based studies have revealed a complex network inside the lumen of both tubules composed of microtubule-inner proteins (MIPs). However, the functions of most MIPs remain unknown. Here, we present single-particle cryo-EM-based analyses of the Tetrahymena thermophila native doublet microtubule and identify 42 MIPs. These data shed light on the evolutionarily conserved and diversified roles of MIPs. In addition, we identified MIPs potentially responsible for the assembly and stability of the doublet outer junction. Knockout of the evolutionarily conserved outer junction component CFAP77 moderately diminishes Tetrahymena swimming speed and beat frequency, indicating the important role of CFAP77 and outer junction stability in cilia beating generation and/or regulation.

Key essential oil components delocalize Candida albicans Kar3p and impact microtubule structure.

BACKGROUND: Treatment of Candida albicans associated infections is often ineffective in the light of resistance, with an urgent need to discover novel antimicrobials. Fungicides require high specificity and can contribute to antifungal resistance, so inhibition of fungal virulence factors is a good strategy for developing new antifungals. OBJECTIVES: Examine the impact of four plant-derived essential oil components (1,8-cineole, α-pinene, eugenol, and citral) on C. albicans microtubules, kinesin motor protein Kar3 and morphology. METHODS: Microdilution assays were used to determine minimal inhibitory concentrations, microbiological assays assessed germ tube, hyphal and biofilm formation, confocal microscopy probed morphological changes and localization of tubulin and Kar3p, and computational modelling was used to examine the theoretical binding of essential oil components to tubulin and Kar3p. RESULTS: We show for the first time that essential oil components delocalize the Kar3p, ablate microtubules, and induce psuedohyphal formation with reduced biofilm formation. Single and double deletion mutants of kar3 were resistant to 1,8-cineole, sensitive to α-pinene and eugenol, but unimpacted by citral. Strains with homozygous and heterozygous Kar3p disruption had a gene-dosage effect for all essential oil components, resulting in enhanced resistance or susceptibility patterns that were identical to that of cik1 mutants. The link between microtubule (αß-tubulin) and Kar3p defects was further supported by computational modeling, showing preferential binding to αß-tubulin and Kar3p adjacent to their Mg2+-binding sites. CONCLUSION: This study highlights how essential oil components interfere with the localization of the kinesin motor protein complex Kar3/Cik1 and disrupt microtubules, leading to their destabilization which results in hyphal and biofilm defects.

Upregulation of ENKD1 disrupts cellular homeostasis to promote lymphoma development.

Diffuse large B cell lymphoma (DLBCL) is a common and aggressive form of B cell lymphoma. Approximately 40% of DLBCL patients are incurable despite modern therapeutic approaches. To explore the molecular mechanisms driving the growth and progression of DLBCL, we analyzed genes with differential expression in DLBCL using the Gene Expression Profiling Interactive Analysis database. Enkurin domain-containing protein 1 (ENKD1), a centrosomal protein-encoding gene, was found to be highly expressed in DLBCL samples compared with normal samples. The phylogenetic analysis revealed that ENKD1 is evolutionarily conserved. Depletion of ENKD1 in cultured DLBCL cells induced apoptosis, suppressed cell proliferation, and blocked cell cycle progression in the G2/M phase. Moreover, ENKD1 expression positively correlates with the expression levels of a number of cellular homeostatic regulators, including Sperm-associated antigen 5, a gene encoding an important mitotic regulator. These findings thus demonstrate a critical function for ENKD1 in regulating the cellular homeostasis and suggest a potential value of targeting ENKD1 for the treatment of DLBCL.

Systematic analysis of microtubule plus-end networks defines EB-cargo complexes critical for mitosis in budding yeast.

Microtubules are ubiquitous cytoskeletal polymers with essential functions in chromosome segregation, intracellular transport, and cellular morphogenesis. End-binding proteins (EBs) form the nodes of intricate microtubule plus-end interaction networks. Which EB binding partners are most critical for cell division and how cells organize a microtubule cytoskeleton in the absence of an EB protein are open questions. Here, we perform a detailed analysis of deletion and point mutants of the budding yeast EB protein Bim1. We demonstrate that Bim1 executes its key mitotic functions as part of two cargo complexes-Bim1-Kar9 in the cytoplasm and Bim1-Bik1-Cik1-Kar3 in the nucleus. The latter complex acts during initial metaphase spindle assembly and supports tension establishment and sister chromatid biorientation. We demonstrate that engineered plus-end targeting of Cik1-Kar3 and overexpression of the microtubule crosslinker Ase1 restore distinct aspects of the bim1Δ spindle phenotype. In addition to defining key Bim1-cargo complexes our study also characterizes redundant mechanisms that allow cells to proliferate in the absence of Bim1.

Nesprin-1 LINC complexes recruit microtubule cytoskeleton proteins and drive pathology in Lmna-mutant striated muscle.

Mutations in LMNA, the gene encoding A-type lamins, cause laminopathies-diseases of striated muscle and other tissues. The aetiology of laminopathies has been attributed to perturbation of chromatin organization or structural weakening of the nuclear envelope (NE) such that the nucleus becomes more prone to mechanical damage. The latter model requires a conduit for force transmission to the nucleus. NE-associated Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes are one such pathway. Using clustered regularly interspaced short palindromic repeats to disrupt the Nesprin-1 KASH (Klarsicht, ANC-1, Syne Homology) domain, we identified this LINC complex protein as the predominant NE anchor for microtubule cytoskeleton components, including nucleation activities and motor complexes, in mouse cardiomyocytes. Loss of Nesprin-1 LINC complexes resulted in loss of microtubule cytoskeleton proteins at the nucleus and changes in nuclear morphology and positioning in striated muscle cells, but with no overt physiological defects. Disrupting the KASH domain of Nesprin-1 suppresses Lmna-linked cardiac pathology, likely by reducing microtubule cytoskeleton activities at the nucleus. Nesprin-1 LINC complexes thus represent a potential therapeutic target for striated muscle laminopathies.

Reduced SPAG17 Expression in Systemic Sclerosis Triggers Myofibroblast Transition and Drives Fibrosis.

Systemic sclerosis (SSc) is a clinically heterogeneous fibrotic disease with no effective treatment. Myofibroblasts are responsible for unresolving synchronous skin and internal organ fibrosis in SSc, but the drivers of sustained myofibroblast activation remain poorly understood. Using unbiased transcriptome analysis of skin biopsies, we identified the downregulation of SPAG17 in multiple independent cohorts of patients with SSc, and by orthogonal approaches, we observed a significant negative correlation between SPAG17 and fibrotic gene expression. Fibroblasts and endothelial cells explanted from SSc skin biopsies showed reduced chromatin accessibility at the SPAG17 locus. Remarkably, mice lacking Spag17 showed spontaneous skin fibrosis with increased dermal thickness, collagen deposition and stiffness, and altered collagen fiber alignment. Knockdown of SPAG17 in human and mouse fibroblasts and microvascular endothelial cells was accompanied by spontaneous myofibroblast transformation and markedly heightened sensitivity to profibrotic stimuli. These responses were accompanied by constitutive TGF-ß pathway activation. Thus, we discovered impaired expression of SPAG17 in SSc and identified, to our knowledge, a previously unreported cell-intrinsic role for SPAG17 in the negative regulation of fibrotic responses. These findings shed fresh light on the pathogenesis of SSc and may inform the search for innovative therapies for SSc and other fibrotic conditions through SPAG17 signaling.

Modeling Study of the Dynamics of Kinesin-14 Molecular Motors.

Kinesin-14s constitute a subfamily of the large superfamily of adenosine triphosphate-dependent microtubule-based motor proteins. Kinesin-14s have the motor domain at the C-terminal end of the peptide, playing key roles during spindle assembly and maintenance. Some of them are nonprocessive motors, whereas others can move processively on microtubules. Here, we take budding yeast Cik1-Kar3 and human HSET as examples to study theoretically the dynamics of the processive kinesin-14 motor moving on the single microtubule under load, the dynamics of the motor coupled with an Ndc80 protein moving on the single microtubule, the dynamics of the motor moving in microtubule arrays, and so on. The dynamics of the nonprocessive Drosophila Ncd motor is also discussed. The studies explain well the available experimental data and, moreover, provide predicted results. We show that the processive kinesin-14 motors can move efficiently in microtubule arrays toward the minus ends, and after reaching the minus ends, they can stay there stably, thus performing the function of organizing the microtubules in the bipolar spindle into polar arrays at the spindle poles.

Integrative transcriptome analysis reveals TEKT2 and PIAS2 involvement in diabetic nephropathy.

Cell heterogeneity has impeded the accurate interpretation of the bulk transcriptome data from patients with diabetic nephropathy (DN). We performed an analysis by integrating bulk and single-cell transcriptome datasets to uncover novel mechanisms leading to DN, especially in the podocytes. Microdissected glomeruli and tubules transcriptome datasets were selected from Gene Expression Omnibus (GEO). Then the consistency between datasets was evaluated. The analysis of the bulk dataset and single-nucleus RNA dataset was integrated to reveal the cell type-specific responses to DN. The candidate genes were validated in kidney tissues from DN patients and diabetic mice. We compared 4 glomerular and 4 tubular datasets and found considerable discrepancies among datasets regarding the deferentially expressed genes (DEGs), involved signaling pathways, and the hallmark enrichment profiles. Deconvolution of the bulk data revealed that the variations in cell-type proportion contributed greatly to this discrepancy. The integrative analysis uncovered that the dysregulation of spermatogenesis-related genes, including TEKT2 and PIAS2, was involved in the development of DN. Importantly, the mRNA level of TEKT2 was negatively correlated with the mRNA levels of NPHS1 (r = -.66, p < .0001) and NPHS2 (r = -.85, p < .0001) in human diabetic glomeruli. Immunostaining confirmed that the expression of TEKT2 and PIAS2 were up-regulated in podocytes of DN patients and diabetic mice. Knocking down TEKT2 resisted high glucose-induced cytoskeletal remodeling and down-regulation of NPHS1 protein in the cultured podocyte. In conclusion, the integrative strategy can help us efficiently use the publicly available transcriptomics resources. Using this approach and combining it with classical research methods, we identified TEKT2 and PIAS2, two spermatogenesis-related genes involved in the pathogenesis of DN. Furthermore, TEKT2 is involved in this pathogenesis by regulating the podocyte cytoskeleton.

Ultrafast Force-Clamp Spectroscopy of Microtubule-Binding Proteins.

Optical trapping has been instrumental for deciphering translocation mechanisms of the force-generating cytoskeletal proteins. However, studies of the dynamic interactions between microtubules (MTs) and MT-associated proteins (MAPs) with no motor activity are lagging. Investigating the motility of MAPs that can diffuse along MT walls is a particular challenge for optical-trapping assays because thermally driven motions rely on weak and highly transient interactions. Three-bead, ultrafast force-clamp (UFFC) spectroscopy has the potential to resolve static and diffusive translocations of different MAPs with sub-millisecond temporal resolution and sub-nanometer spatial precision. In this report, we present detailed procedures for implementing UFFC, including setup of the optical instrument and feedback control, immobilization and functionalization of pedestal beads, and preparation of MT dumbbells. Example results for strong static interactions were generated using the Kinesin-7 motor CENP-E in the presence of AMP-PNP. Time resolution for MAP-MT interactions in the UFFC assay is limited by the MT dumbbell relaxation time, which is significantly longer than reported for analogous experiments using actin filaments. UFFC, however, provides a unique opportunity for quantitative studies on MAPs that glide along MTs under a dragging force, as illustrated using the kinetochore-associated Ska complex.

Defects in division plane positioning in the root meristematic zone affect cell organization in the differentiation zone.

Cell-division-plane orientation is critical for plant and animal development and growth. TANGLED1 (TAN1) and AUXIN-INDUCED IN ROOT CULTURES 9 (AIR9) are division-site-localized microtubule-binding proteins required for division-plane positioning. The single mutants tan1 and air9 of Arabidopsis thaliana have minor or no noticeable phenotypes, but the tan1 air9 double mutant has synthetic phenotypes including stunted growth, misoriented divisions and aberrant cell-file rotation in the root differentiation zone. These data suggest that TAN1 plays a role in non-dividing cells. To determine whether TAN1 is required in elongating and differentiating cells in the tan1 air9 double mutant, we limited its expression to actively dividing cells using the G2/M-specific promoter of the syntaxin KNOLLE (pKN:TAN1-YFP). Unexpectedly, in addition to rescuing division-plane defects, expression of pKN:TAN1-YFP rescued root growth and cell file rotation defects in the root-differentiation zone in tan1 air9 double mutants. This suggests that defects that occur in the meristematic zone later affect the organization of elongating and differentiating cells.

Lrwd1 impacts cell proliferation and the silencing of repetitive DNA elements.

LRWD1, also known as ORCA, is a nuclear protein functioning in multiple biological processes. Using its WD40 domain LRWD1 interacts with repressive histone marks and maintains the silencing of heterochromatin regions in mammalian cells. ORCA also associates with the origin recognition complex (ORC) and facilitates prereplication complex formation at late-replicating origins. However, whether LRWD1 plays a role during development and the functional significance of LRWD1 in vivo remains largely unknown. Using gene-trap approach we generated Lrwd1 knockout mice and examined the expression of Lrwd1 during embryonic development. We found that Lrwd1 is ubiquitously expressed in the majority of the developing mouse embryo. Depletion of LRWD1 did not affect embryonic development but the postnatal growth of the homozygous mutants is retarded. In vitro cultured mouse embryonic fibroblasts (MEFs) depleted of LRWD1 displayed a reduced proliferation compared to wild type cells. We also showed that the knockout of Lrwd1 in MEFs increased the expression of the epigenetically silenced repetitive elements but with minimal effect on the expression of protein coding genes. Together, these results suggest that LRWD1 plays an important, but not essential, role in postnatal development by regulating cell proliferation likely through modulating DNA replication.

ExSTED microscopy reveals contrasting functions of dopamine and somatostatin CSF-c neurons along the lamprey central canal.

Cerebrospinal fluid-contacting (CSF-c) neurons line the central canal of the spinal cord and a subtype of CSF-c neurons expressing somatostatin, forms a homeostatic pH regulating system. Despite their importance, their intricate spatial organization is poorly understood. The function of another subtype of CSF-c neurons expressing dopamine is also investigated. Imaging methods with a high spatial resolution (5-10 nm) are used to resolve the synaptic and ciliary compartments of each individual cell in the spinal cord of the lamprey to elucidate their signalling pathways and to dissect the cellular organization. Here, light-sheet and expansion microscopy resolved the persistent ventral and lateral organization of dopamine- and somatostatin-expressing CSF-c neuronal subtypes. The density of somatostatin-containing dense-core vesicles, resolved by stimulated emission depletion microscopy, was shown to be markedly reduced upon each exposure to either alkaline or acidic pH and being part of a homeostatic response inhibiting movements. Their cilia symmetry was unravelled by stimulated emission depletion microscopy in expanded tissues as sensory with 9 + 0 microtubule duplets. The dopaminergic CSF-c neurons on the other hand have a motile cilium with the characteristic 9 + 2 duplets and are insensitive to pH changes. This novel experimental workflow elucidates the functional role of CSF-c neuron subtypes in situ paving the way for further spatial and functional cell-type classification.