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Results for “cell biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 577 records · Page 32Linked to original sources

bioRxiv2025

Nucleophagy removes cytotoxic trapped PARP1

Poly (ADP-Ribose) Polymerase inhibitors (PARPi) induce cytotoxicity in homologous recombination repair (HRR)-deficient cancers by causing PARP1 to become trapped on chromatin, resulting in irreparable replication-associated DNA damage. Although increased clearance of trapped PARP1 from chromatin reduces the sensitivity of cancer cells to PARPi, details surrounding this process remain unclear. PARPi exposure is known to cause increased autophagy flux, whilst autophagy inhibition can hypersensitise cells to PARPi. Using various biochemical, cell biological and live imaging-based assays, we found that trapped PARP1 is cleared by nucleophagy, the selective autophagy of nuclear substrates. Specifically, the nucleophagy of trapped PARP1 was orchestrated by the selective autophagy receptor TEX264 and its partner segregase p97/VCP. TEX264 mediates this process by directly interacting with trapped PARP1, thus bridging PARP1 to the autophagosomal resident protein LC3 for processing via autophagy. Impeding this process, either chemically or genetically, heightened PARP1 trapping, leading to accumulation of protein aggregates, replication-associated DNA damage and cell lethality, re-sensitising PARPi-resistant cells to various PARPi. In conclusion, we show that nucleophagy acts in a cytoprotective manner to directly target PARPi-induced trapped PARP1 for degradation.

cell biology↗

bioRxiv2020

Both adaptive immunity and IL-1R1 dependent signals improve clearance of cytosolic virulent mycobacteria in vivo

Mycobacterium tuberculosis infections claim more than a million lives each year and better treatments or vaccines are required. A crucial pathogenicity factor is translocation from the phago-lysosomes to the cytosol upon phagocytosis by macrophages. The translocation from the phago-lysosome into the cytosol is an ESX-1 dependent process as previously shown in vitro. Here we show that in vivo, mycobacteria also translocate to the cytosol but mainly when host immunity is compromised. We observed only low numbers of cytosolic bacilli in mice, armadillo, zebrafish and patient material infected with M. tuberculosis, M. marinum or M. leprae. In contrast, when innate or adaptive immunity was compromised, as in SCID or IL-1R1 deficient mice, a significant number of cytosolic M. tuberculosis bacilli were detected in lungs of infected mice. Taken together, M. tuberculosis infection is controlled by adaptive immune responses as well as IL-1R1-mediated signals that result in clearance of cells containing cytosolic mycobacteria in vivo. ImportanceFor decades, Mycobacterium tuberculosis is one of the deathliest pathogens known. Despite infecting approximately one third of the human population, no effective treatment or vaccine is available. A crucial pathogenicity factor is the subcellular localization, as M. tuberculosis can translocate from the phago-lysosome to the cytosol in macrophages. The situation in vivo is more complicated. In this study we establish that high level cytosolic escape of mycobacteria can indeed occur in vivo, but mainly when host resistance is compromised. The IL-1 pathway is crucial for the control of the number of cytosolic mycobacteria. The establishment that immune signals result in clearance of cells containing cytosolic mycobacteria, connects two important fields: cell-biology and immunology which is vital for the understanding of the pathology of M. tuberculosis.

cell biology↗

bioRxiv2022

Deep neural network automated segmentation of cellular structures in volume electron microscopy

Recent advances in automated segmentation using deep neural network models allow identification of intracellular structures. This study describes a new pipeline to train a convolutional neural network for rapid and efficient detection of structures of wide range in size and complexity. AbstractThree-dimensional electron microscopy is an important imaging modality in contemporary cell biology. Identification of intracellular structures is laborious and time-consuming, however, and impairs effective use of a potentially powerful tool. Resolving this bottleneck is therefore a critical next step in frontier biomedical imaging. We describe Automated Segmentation of intracellular substructures in Electron Microscopy (ASEM), a new pipeline to train a convolutional neural network to detect structures of wide range in size and complexity. We obtain for each structure a dedicated model based on a small number of sparsely annotated ground truth annotations from only one or two cells. To improve model generalization to different imaging conditions, we developed a rapid, computationally effective strategy to refine an already trained model by including a few additional annotations. We show the successful automated identification of mitochondria, Golgi apparatus, endoplasmic reticulum, nuclear pore complexes, caveolae, clathrin coated pits and coated vesicles in cells imaged by focused ion beam scanning electron microscopy with quasi-isotropic resolution. ASEM enabled us to uncover a wide range of membrane-nuclear pore diameters within a single cell and to derive morphological metrics from clathrin coated pits and vesicles at all stages of maturation consistent with the classical constant-growth assembly model.

cell biology↗

bioRxiv2025

MAFA Phosphorylation Controls Beta-Cell Identity and Sex-Specific Pancreatic Disease Outcomes

Mafa is a critical transcription factor in pancreatic beta-cell biology, orchestrating insulin expression in response to glucose elevations. As a member of the large MAF protein family, MAFAs stability and activity are intricately regulated by GSK3-mediated phosphorylation. To decipher the functional roles of these phosphorylations, we engineered knock-in mice (Mafa4A/+) in which MAFA is rendered non-phosphorylatable. In all Mafa4A/+ animals, MAFA stability was markedly enhanced. Under high-fat diet (HFD) conditions, Mafa4A/+ males rapidly developed glucose intolerance, which was attributed to impaired glucose-stimulated insulin secretion. Bulk RNA sequencing revealed disrupted beta- cell identity, characterized by increased expression of MODY-associated genes and a delta-cell signature, suggesting beta-to-delta cell reprogramming, a hypothesis supported by lineage- tracing experiments. Conversely, Mafa4A/+ females exhibited hypoglycemia and, with age, developed pronounced inflammatory cystic ducts including mucinous cystic neoplasms (MCNs). Strikingly, MAFA protein was also detected in MCN biopsies from female patients, linking our findings to human pathology. Our results unveil a sex-biased impact of GSK3-mediated MAFA phosphorylation. The male phenotype closely parallels the MODY-like diabetes observed in patients with MAFA S64F mutations, implicating defective phosphorylation in disease etiology. The emergence of MCNs in female mice suggests a novel role for MAFA stability or mutations in the pathogenesis of these enigmatic neoplasms, providing a fresh molecular hypothesis with clinical relevance.

cell biology↗

bioRxiv2016

TriPer, an optical probe tuned to the endoplasmic reticulum, tracks H2O2 consumption by glutathione

The fate of H2O2 in the endoplasmic reticulum (ER) has been inferred indirectly from the activity of ER localized thiol oxidases and peroxiredoxins, in vitro, and the consequences of their genetic manipulation, in vivo. Here we report on the development of TriPer, a vital optical probe sensitive to changes in the concentration of H2O2 in the thiol-oxidizing environment of the ER. Consistent with the hypothesized contribution of oxidative protein folding to H2O2 production, ER-localized TriPer detected an increase in the luminal H2O2 signal upon induction of pro-insulin (a disulfide bonded protein of pancreatic {beta}-cells), which was attenuated by the ectopic expression of catalase in the ER lumen. Interfering with glutathione production in the cytosol by buthionine sulfoximine (BSO) or enhancing its localized destruction by expression of the glutathione-degrading enzyme ChaC1 in lumen of the ER, enhanced further the luminal H2O2 signal and eroded {beta}-cell viability. Tracking ER H2O2 in live cells points to an unanticipated role for glutathione in H2O2 turnover.\n\nSignificance statementThe presence of millimolar glutathione in the lumen of the endoplasmic reticulum has been difficult to understand purely in terms of modulation of protein-based disulphide bond formation in secreted proteins. Over the years hints have suggested that glutathione might have a role in reducing the heavy burden of hydrogen peroxide (H2O2) produced by the luminal enzymatic machinery for disulphide bond formation. However, limitations in existing in vivo H2O2 probes have rendered them all but useless in the thiol-oxidizing ER, precluding experimental follow-up of glutathiones role ER H2O2 metabolism.\n\nHere we report on the development and mechanistic characterization of an optical probe, TriPer that circumvents the limitations of previous sensors by retaining specific responsiveness to H2O2 in thiol-oxidizing environments. Application of this tool to the ER of an insulin-producing pancreatic b-cells model system revealed that ER glutathione antagonizes locally-produced H2O2 resulting from the oxidative folding of pro-insulin.\n\nThis study presents an interdisciplinary effort intersecting cell biology and chemistry: An original redox chemistry concept leading to development of a biological tool, broadly applicable for in vivo studies of H2O2 metabolism in the ER. More broadly, the concept developed here sets a precedent for applying a tri-cysteine relay system to discrimination between various oxidative reactants, in complex redox milieux.

cell biology↗

bioRxiv2023

A fitness landscape instability governs the morphological diversity of tip-growing cells

Cellular morphology affects many aspects of cellular and organismal physiology. This makes it challenging to understand the evolutionary basis for specific morphologies since the various facets of cellular physiology may exert competing selective pressures on this trait. The influence of these pressures, moreover, will depend on the mechanisms of cellular morphogenesis. To address this problem, we combined experimental cell biology with mechanics-based theory to analyze the morphological diversity of tip-growing cells from across the tree of life. We discovered that an instability in the convergent mechanism of "inflationary" growth shared by these cells leads directly to a bifurcation in their fitness landscape, which imposes a strong global constraint on their morphologies. Additionally, we found that co-selection for cell size and elongation rate explains variation among observable morphologies. This analysis rationalizes the morphology - and provides quantitative insight into the ecology - of an enormous diversity of important fungal, plant, protistan, and bacterial systems. Additionally, our study elucidates a fundamental principle of evolutionary-developmental biology that would be difficult to rigorously demonstrate in more complex systems.

cell biology↗

bioRxiv2024

SpotMAX: a generalist framework for multi-dimensional automatic spot detection and quantification

The analysis of spot-like structures is a widespread task in microscopy-based cell biology. Existing solutions are typically specific to single applications and do not use multi-dimensional information from 5D datasets. Therefore, experimental scientists often resort to subjective manual annotation. Here, we present SpotMAX, a generalist AI-driven framework for automated spot detection and quantification. SpotMAX leverages the full scope of multi-dimensional datasets with an easy-to-use interface and an embedded framework for cell segmentation and tracking. SpotMAX outperforms state-of-the-art tools, and in some cases, even expert human annotators. We applied SpotMAX across diverse experimental questions, ranging from meiotic crossover events in C. elegans to mitochondrial DNA dynamics in S. cerevisiae and telomere length in mouse stem cells, leading to new biological insights. With its flexibility in integrating AI workflows, we anticipate that SpotMAX will become the standard for spot analysis in microscopy data. Source code: https://github.com/SchmollerLab/SpotMAX

cell biology↗

bioRxiv2025

PIKI-1, a class II phosphatidylinositol 3-kinase, functions in endocytic trafficking

Membrane trafficking, including endocytosis and exocytosis, is a complex process that is coordinated by trafficking-associated proteins, cargo molecules, the cytoskeleton, and membrane lipid composition. The NIMA-related kinases NEKL-2 (human NEK8/9) and NEKL-3 (human NEK6/7) are conserved regulators of membrane trafficking in Caenorhabditis elegans and are required for successful molting. Through a genetic approach, we isolated reduction-of-function mutations in piki-1 that suppress nekl-associated molting defects. piki-1 encodes the sole predicted C. elegans Class II phosphatidylinositol 3-kinase (PI3Ks), an understudied class of lipid modifiers that contribute to the production of phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). Using a set of genetically encoded lipid sensors, we found that PIKI-1 was responsible for the production of PI(3,4)P2 in the C. elegans epidermis but played only a minor role in the control of PI(3)P levels. Consistent with this, both PI(3,4)P2 and PIKI-1 colocalized to early endosomes, and reduction of PIKI-1 function strongly affected early endosomal morphology and protein composition. Additionally, reduced PIKI-1 function led to excess tubulation of endosomal compartments associated with recycling or the degradation of cellular debris. In contrast to previous studies using mammalian cell culture, PIKI-1 was largely dispensable for clathrin-mediated endocytosis in the context of the worm epidermis, which is a polarized epithelium. Notably, reduction of PIKI-1 function strongly mitigated defects in early endosomes associated with the depletion of NEKL-2. We propose that reduction of PIKI-1 function may suppress nekl molting defects by partially restoring endocytic trafficking within specific compartments, including the early endosome. We also show that inhibition of the PI(3,4)P2-binding protein HIPR-1 (HIP1/HIPR1) suppresses nekl molting defects, suggesting that reduced PI(3,4)P2 levels alter endosomal protein recruitment in a manner that antagonizes NEKL-2 function. Author summaryThe uptake of materials from outside the cell and their subsequent delivery to specific intracellular locations are essential for cell function and survival. Two of the mechanisms that control this complex intracellular pathway involve the modification of proteins and of lipids, processes that are highly conserved across species. In this study, we used the model organism Caenorhabditis elegans, which is highly amenable to cell biological and genetic approaches, to establish a novel connection between these two regulatory mechanisms and demonstrate the importance of lipid modifications in maintaining the normal functioning of intracellular transport. Our results also provide insights into the fundamental cellular functions of proteins associated with human disease including cancer and metabolic disease.

cell biology↗

bioRxiv2025

PyCLM: programming-free, closed-loop microscopy for real-time measurement, segmentation, and optogenetic stimulation

In cell biology, optical techniques are increasingly used to measure cells internal states (biosensors) and to stimulate cellular responses (optogenetics). Yet the design of all-optical experiments is often manual: a pre-determined stimulus pattern is applied to cells, biosensors are measured over time, and the resulting data is processed off-line. With the advent of machine learning for segmentation and tracking, it becomes possible to envision closed-loop experiments where real-time information about cells positions and states are used to dynamically determine optogenetic stimuli to alter or control their behavior. Here, we develop PyCLM, a Python-based suite of tools to enable real-time measurement, image segmentation, and optogenetic control of thousands of cells per experiment. PyCLM is designed to be as simple for the end user as possible, and multipoint experiments can be set up that combine a wide variety of imaging, image processing, and stimulation modalities without any programming. We showcase PyCLM on diverse applications: studying the effect of epidermal growth factor receptor activity waves on epithelial tissue movement, simultaneously stimulating [~]1,000 single cells to guide tissue flows, and performing real-time feedback control of cell-to-cell fluorescence heterogeneity. This tool will enable the next generation of dynamic experiments to probe cell and tissue properties, and provides a first step toward precise control of cell states at the tissue scale.

systems biology↗

bioRxiv2019

Neural differentiation is increased by GSK-3β inhibition and decreased by tankyrase inhibition in human neural precursor cells

Glycogen synthase kinase-3{beta} (GSK-3{beta}) and tankyrase-1/2 (TANK) are two enzymes known to play multiple roles in cell biology, including regulation of proliferation, differentiation and metabolism. Both of them act on the canonical Wnt/{beta}-Catenin pathway, but are also involved in many other independent intracellular mechanisms. More importantly, GSK-3{beta} and TANK have been shown to play crucial roles in different diseases, including cancer and neurological disorders. The GSK-3{beta}-inhibitor CHIR and the TANK-inhibitor XAV are two pyrimidine molecules, holding high potential as possible therapeutic drugs. However, their effect on neural tissue is poorly understood. In this study, we tested the effects of CHIR and XAV on human neural precursor cells (hNPCs) derived from human embryonic stem cells. We found that CHIR-mediated inhibition of GSK-3{beta} promotes neural differentiation. In contrast, XAV-mediated inhibition of TANK leads to de-differentiation. These results highlight the relative importance of these two enzymes in determining the neurodevelopmental status of hNPCs. Furthermore, they shed light on the roles of Wnt signaling during early human neurogenesis.

cell biology↗

bioRxiv2022

The Intrinsically Disordered Region of Coronins Fine-tunes Oligomerization and Actin Polymerization

Coronins are highly conserved actin-binding proteins (ABPs) in the eukaryotic kingdom for polymerizing actin cytoskeleton. The biochemical activity of coronins is primarily mediated by the structural N-terminal {beta}-propeller and the C-terminal helical coiled-coil (CC) domains, but less is known about the function of a middle nonconserved region, the "unique region (UR)". The coronin UR is an intrinsically disordered region (IDR). Herein, we demonstrate that the low complexity of the UR is a conserved signature of the coronin protein family, and the UR/IDR exhibits a striking evolutionary correlated pattern associated with sequence length. By analyzing the role of the IDR in coronins via coarse-grained simulations, we reveal that evolutionary selection of IDR length is coupled with the oligomerization of IDR-containing proteins (IDPs) to provide optimal functional output. By integrating biochemical and cell biology experiments and protein engineering, we found that the IDR regulates Crn1 biochemical activity, both in vivo and in vitro, by fine-tuning CC domain oligomerization and maintaining Crn1 in a tetrameric state. The IDR-guided optimization of Crn1 oligomerization is critical for Arp2/3-mediated actin polymerization.

cell biology↗

bioRxiv2021

A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C driven phosphoinositide conversion

Polarized trafficking is necessary for the development of eukaryotes and is regulated by a conserved molecular machinery. Late steps of cargo delivery are mediated by the exocyst complex, which integrates lipid and protein components to tether vesicles for plasma membrane fusion. However, the molecular mechanisms of this process are poorly defined. Here, we reconstitute functional octameric human exocyst, demonstrating the basis for holocomplex coalescence and biochemically stable subcomplexes. We determine that each subcomplex independently binds to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which is minimally sufficient for membrane tethering. Through reconstitution and epithelial cell biology experiments, we show that Arf6-mediated recruitment of the lipid kinase PIP5K1C rapidly converts phosphatidylinositol 4-phosphate (PI(4)P) to PI(4,5)P2, driving exocyst recruitment and membrane tethering. These results provide a molecular mechanism of exocyst-mediated tethering and a unique functional requirement for phosphoinositide signaling on latestage vesicles in the vicinity of the plasma membrane.

cell biology↗

bioRxiv2024

Charting the landscape of cytoskeletal diversity in microbial eukaryotes

Microbial eukaryotes are small and often resistant to standard labelling and imaging techniques, and therefore remain understudied - despite their critical ecological importance - with the exception of a few established models. Here, we use Ultrastructure Expansion Microscopy (U-ExM) to carry out high-resolution volumetric imaging of over 200 cultured planktonic eukaryotes across major lineages. By combining U-ExM with pan- and specific immuno-labelling, we reveal novel microtubule and centrin-containing elements and assign molecular identities to enigmatic cytoskeletal structures observed previously only by electron microscopy. Our investigation represents the first systematic survey of the extensive cytoskeletal diversity on display across the eukaryotic tree, including the major species groups of dinoflagellates, haptophytes, ciliates, euglenids, cryptomonads, and green algae. Our U-ExM approach extends to mixed environmental samples, paving the way for environmental cell biology at ultrastructural resolution and unprecedented scale, a crucial step towards understanding and protecting complex ecosystems in the face of biodiversity loss.

cell biology↗

bioRxiv2024

Image Correlation Spectroscopy is a Robust Tool to Quantify Cellular DNA Damage Response

The DNA Damage response (DDR) is both essential and highly complex. Evaluating the DDR is a critical aspect of cell biology. Counting DNA damage foci is one of the most common approaches to study the DDR. Yet, quantification of protein foci suffers from experimental limitations, subjectivity of analysis and is restricted to a handful of the hundreds of DDR proteins. Here we apply image correlation spectroscopy (ICS) to quantify the local clustering at sites of DNA damage directly. We found that ICS outperformed foci counting of traditional DDR markers and enabled quantification of other markers without the complex labeling procedures that are otherwise required. ICS analysis also provided insight into DDR protein recruitment that was previously undetectable. Further expansion incorporating analysis to cell cycle classification demonstrates a rapid, non-biased approach to fully study the DNA damage response within cells. ICS analysis presents an objective, quantitative image analysis technique to study the DNA damage response in unaltered cells that we expect will significantly enhance quantitative DNA damage response research.

cell biology↗

bioRxiv2018

Lineage tracing on transcriptional landscapes links state to fate during differentiation

A challenge in stem cell biology is to associate molecular differences among progenitor cells with their capacity to generate mature cell types. Though the development of single cell assays allows for the capture of progenitor cell states in great detail, these assays cannot definitively link cell states to their long-term fate. Here, we use expressed DNA barcodes to clonally trace single cell transcriptomes dynamically during differentiation and apply this approach to the study of hematopoiesis. Our analysis identifies functional boundaries of cell potential early in the hematopoietic hierarchy and locates them on a continuous transcriptional landscape. We reconstruct a developmental hierarchy showing separate ontogenies for granulocytic subtypes and two routes to monocyte differentiation that leave a persistent imprint on mature cells. Finally, we use our approach to benchmark methods of dynamic inference from single-cell snapshots, and provide evidence of strong early fate biases dependent on cellular properties hidden from single-cell RNA sequencing.

systems biology↗

bioRxiv2021

Talin mechanosensitivity is modulated by a direct interaction with cyclin-dependent kinase-1

Talin is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behaviour of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronised phosphorylation of a large number of protein targets. CDK1 activity also maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesiveness that are required for successful completion of mitosis. Using a combination of biochemical, structural and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin-mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8 through the use of recombinant fragments. Talin also contains a consensus CDK1 phosphorylation motif centred on S1589; a site that was phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of KANK and weakened the mechanical response of the region, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator, CDK1, to the primary integrin effector, talin, therefore provides a primordial solution for coupling the cell proliferation and cell adhesion machineries, and thereby enables microenvironmental control of cell division in multicellular organisms. SummaryThe direct binding of the master cell cycle regulator, CDK1, to the primary integrin effector, talin, provides a primordial solution for coupling the cell proliferation and cell adhesion machineries, and thereby enables microenvironmental control of cell division.

cell biology↗

bioRxiv2026

BARTsc identifies key transcriptional regulators from single-cell omics data

Inference of transcriptional regulatory mechanisms from single-cell (sc) omics data, such as scRNA-seq, scATAC-seq, and scMultiome, remains an important problem in single-cell biology and functional genomics. Most existing methods for predicting functional transcriptional regulators (TRs) from single-cell data rely on co-expression between regulator and target genes and/or sequence motif enrichment, holding inherent limitations. Here, we present BARTsc, a computational method that accurately predicts functional TRs from clustered single-cell omics data by leveraging a large collection of public ChIP-seq profiles. BARTsc implements a novel framework to infer a cis-regulatory profile from differential genomic features from either unimodal (RNA or ATAC) or bimodal (scMultiome) single-cell profiling data and identify TRs whose binding profiles most associate with the cis-regulatory profile. BARTsc can quantify TR activity across cell clusters and predict key regulators for each cell cluster. We demonstrate that BARTsc can successfully identify active TRs in each cell type and cell-type-defining key regulators across diverse biological systems, including mouse cortex, human peripheral blood mononuclear cells (PBMCs), and human pancreatic ductal adenocarcinoma (PDAC). Using a generative-AI-assisted, literature-supported collection of cell-type key regulators as benchmarks, we show that BARTsc consistently outperforms existing state-of-the-art methods. We apply BARTsc to identify critical regulators in PDAC, including NEFLA, a novel PDAC key regulator, and validate its function in pancreatic tumor proliferation by experiments. As a robust and versatile computational method, BARTsc provides deeper insights into cell-type-specific regulatory programs, facilitating the discovery of key regulators across diverse biological systems.

bioinformatics↗

bioRxiv2018

Tetrahymena RIB72A and RIB72B are Microtubule Inner Proteins in the ciliary doublet microtubules

Doublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia and flagella. In contrast to dynamic cytoplasmic microtubules, their luminal surface is coated with regularly arranged Microtubule Inner Proteins (MIPs). However, the protein composition and biological function(s) of MIPs remain poorly understood. Using genetic, biochemical and imaging techniques we identified Tetrahymena RIB72A and RIB72B proteins as ciliary MIPs. Fluorescence imaging of tagged RIB72A and RIB72B showed that both proteins co-localize to Tetrahymena cilia and basal bodies, but assemble independently. Cryo-electron tomography of RIB72A and/or RIB72B knockout strains revealed major structural defects in the ciliary A-tubule involving MIP1, MIP4 and MIP6 structures. The defects of individual mutants were complementary in the double mutant. All mutants had reduced swimming speed and ciliary beat frequencies, and high-speed video imaging revealed abnormal highly curved cilia during power stroke. Our results show that RIB72A and RIB72B are crucial for the structural assembly of ciliary A-tubule MIPs and are important for proper ciliary motility.\n\nSUMMARYMicrotubule Inner Proteins (MIPs) bind to the luminal surface of highly stable microtubules. Combining cell biology and cryo-electron tomography, Stoddard et al. show that RIB72A and RIB72B are conserved MIPs in ciliary doublet microtubules and that they are important for proper ciliary motility.

cell biology↗