Microtubules are dynamic cytoskeletal polymers, and their organization and stability are tightly regulated by numerous cellular factors. While regulatory proteins controlling the formation of interphase microtubule arrays and mitotic spindles have been extensively studied, the biochemical mechanisms responsible for generating stable microtubule cores of centrioles and cilia are poorly understood. Here, we used in vitro reconstitution assays to investigate microtubule-stabilizing properties of CSPP1, a centrosome and cilia-associated protein mutated in the neurodevelopmental ciliopathy Joubert syndrome. We found that CSPP1 preferentially binds to polymerizing microtubule ends that grow slowly or undergo growth perturbations and, in this way, resembles microtubule-stabilizing compounds such as taxanes. Fluorescence microscopy and cryo-electron tomography showed that CSPP1 is deposited in the microtubule lumen and inhibits microtubule growth and shortening through two separate domains. CSPP1 also specifically recognizes and stabilizes damaged microtubule lattices. These data help to explain how CSPP1 regulates the elongation and stability of ciliary axonemes and other microtubule-based structures.

MOTIVATION: Biological studies of dynamic processes in living cells often require accurate particle tracking as a first step toward quantitative analysis. Although many particle tracking methods have been developed for this purpose, they are typically based on prior assumptions about the particle dynamics, and/or they involve careful tuning of various algorithm parameters by the user for each application. This may make existing methods difficult to apply by non-expert users and to a broader range of tracking problems. Recent advances in deep-learning techniques hold great promise in eliminating these disadvantages, as they can learn how to optimally track particles from example data. RESULTS: Here, we present a deep-learning-based method for the data association stage of particle tracking. The proposed method uses convolutional neural networks and long short-term memory networks to extract relevant dynamics features and predict the motion of a particle and the cost of linking detected particles from one time point to the next. Comprehensive evaluations on datasets from the particle tracking challenge demonstrate the competitiveness of the proposed deep-learning method compared to the state of the art. Additional tests on real-time-lapse fluorescence microscopy images of various types of intracellular particles show the method performs comparably with human experts. AVAILABILITY AND IMPLEMENTATION: The software code implementing the proposed method as well as a description of how to obtain the test data used in the presented experiments will be available for non-commercial purposes from https://github.com/yoyohoho0221/pt_linking. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Microtubule-dependent organization of membranous organelles occurs through motor-based pulling and by coupling microtubule dynamics to membrane remodeling. For example, tubules of endoplasmic reticulum (ER) can be extended by kinesin- and dynein-mediated transport and through the association with the tips of dynamic microtubules. The binding between ER and growing microtubule plus ends requires End Binding (EB) proteins and the transmembrane protein STIM1, which form a tip-attachment complex (TAC), but it is unknown whether these proteins are sufficient for membrane remodeling. Furthermore, EBs and their partners undergo rapid turnover at microtubule ends, and it is unclear how highly transient protein-protein interactions can induce load-bearing processive motion. Here, we reconstituted membrane tubulation in a minimal system with giant unilamellar vesicles, dynamic microtubules, an EB protein, and a membrane-bound protein that can interact with EBs and microtubules. We showed that these components are sufficient to drive membrane remodeling by three mechanisms: membrane tubulation induced by growing microtubule ends, motor-independent membrane sliding along microtubule shafts, and membrane pulling by shrinking microtubules. Experiments and modeling demonstrated that the first two mechanisms can be explained by adhesion-driven biased membrane spreading on microtubules. Optical trapping revealed that growing and shrinking microtubule ends can exert forces of ∼0.5 and ∼5 pN, respectively, through attached proteins. Rapidly exchanging molecules that connect membranes to dynamic microtubules can thus bear a sufficient load to induce membrane deformation and motility. Furthermore, combining TAC components and a membrane-attached kinesin in the same in vitro assays demonstrated that they can cooperate in promoting membrane tubule extension.