Microtubule (MT) dynamics is tightly regulated by microtubule-associated proteins (MAPs) and various post-translational modifications (PTMs) of tubulin. Here, we introduce OligoMT and OligoTIP as genetically encoded oligomeric MT binders designed for real-time visualization and manipulation of MT behaviors within living cells. OligoMT acts as a reliable marker to label the MT cytoskeleton, while OligoTIP allows for live monitoring of the growing MT plus-ends. These engineered MT binders have been successfully utilized to label the MT network, monitor cell division, track MT plus-ends, and assess the effect of tubulin acetylation on the MT stability at the single-cell level. Moreover, OligoMT and OligoTIP can be repurposed as biosensors for quantitative assessment of drug actions and for reporting enzymatic activity. Overall, these engineered MT binders hold promise for advancing the mechanistic dissection of MT biology and have translational applications in cell-based high-throughput drug discovery efforts.

The microtubule-kinesin biomolecular motor system, which is vital for cellular function, holds significant promise for nanotechnological applications. In vitro gliding assays have demonstrated the ability to transport microcargo by propelling microtubules across kinesin-coated surfaces. However, the uncontrolled directional motion of microtubules has posed significant challenges, limiting the system\'s application for precise cargo delivery. Microfluidic devices provide a means to direct microtubule movement through their geometric features. Norland Optical Adhesive (NOA) is valued for its mold-free application in microfluidic device fabrication; however, microtubules often climb up channel walls, limiting controlled movement. In this study, a surface passivation method for NOA is introduced, using polyethylene glycol via a thiol-ene click reaction. This technique significantly improved the directional control and concentration of microtubules within NOA microchannels. This approach presents new possibilities for the precise application of biomolecular motors in nanotechnology, enabling advancements in the design of microfluidic systems for complex biomolecular manipulations.

The study of microtubules arrangements and dynamics during axon outgrowth and pathfinding has gained scientific interest during the last decade, and numerous technical resources for its visualization and analysis have been implemented. In this chapter, we describe the cell culture protocols of embryonic cortical and retinal neurons, the methods for transfecting them with fluorescent reporters of microtubule polymerization, and the procedures for time-lapse imaging and quantification in order to study microtubule dynamics during axon morphogenesis.

Microtubule-based chemotherapeutics, primarily Taxane-derived agents are still used as the major live-saving agents, yet have several side effects including serious loss of immune cells, bone density etc. which lowers the quality of life. This imposes the need to understand the effects of these agents on Mesenchymal Stem Cells (MSCs) in details. In this work we demonstrate that Taxol and Nocodazole affects the endogenous expression of TRPV1, a non-selective cation channel in MSCs. These agents also affect the status of polymerized Actin as well as Tyrosinated-tubulin, basal cytosolic Ca2+ and mitochondrial membrane potential (ΔΨm). Notably, pharmacological modulation of TRPV1 by Capsaicin or Capsazepine can also alter the above-mentioned parameters in a context-dependent manner. We suggest that endogenous expression of TRPV1 and pharmacological modulation of TRPV1 can be utilized to rescue some of these parameters effectively. These findings may have significance in the treatments and strategies with Microtubule-based chemotherapeutics and stem-cell based therapy.

Over the years immunotherapy has demonstrably improved the field of cancer treatment. However, achieving long-term survival for colorectal cancer (CRC) patients remains a significant unmet need. Combination immunotherapies incorporating targeted drugs like MEK or multi-kinase inhibitors have offered some palliative benefit. Nevertheless, substantial gaps remain in the current therapeutic armamentarium for CRC. In recent years, there has been a surge of interest in exploring novel treatment strategies, including the application of light-activated drugs in conjunction with optical devices. This approach holds promise for achieving localized and targeted delivery of cytotoxic agents, such as microtubule-targeting drugs, directly to cancerous cells within the colon.

Immunotactoid glomerulopathy (ITG) is a rare glomerular disease that typically presents with proteinuria, hematuria, and kidney dysfunction. A kidney biopsy is essential to establish the diagnosis of ITG. ITG is characterized by glomerular electron-dense immunoglobulin deposits with hollow-cored microtubules. ITG is classified as either monoclonal or polyclonal based on immunofluorescence staining of the immunoglobulin deposits. Monoclonal ITG is associated with an underlying hematologic disorder in two-thirds of the cases, lymphoma and plasma cell dyscrasias being the most common. Polyclonal ITG is associated with autoimmune diseases but can be seen with hematologic disorders and chronic infections. Due to the preponderance of hematologic disorders in both monoclonal and polyclonal ITG, a thorough hematologic workup must be performed in all cases of ITG. In monoclonal ITG with a detectable clone, clone-directed therapy is administered to achieve hematologic remission, as the renal response is highly dependent on the hematologic response. In clone-negative monoclonal ITG, anti-B cell therapy is often used as a first-line therapy. Management of polyclonal ITG without an underlying hematologic disorder is poorly defined. Compared to monoclonal ITG, patients with polyclonal ITG have a higher risk of progression to end-stage kidney disease. Recurrence of ITG following kidney transplantation is common and is often associated with hematologic relapse.

Gametes are produced via meiosis, a specialized cell division associated with frequent errors that cause birth defects and infertility. Uniquely in meiosis I, homologous chromosomes segregate to opposite poles, usually requiring their linkage by chiasmata, the products of crossover recombination.1 The spindle checkpoint delays cell-cycle progression until all chromosomes are properly attached to microtubules,2 but the steps leading to the capture and alignment of chromosomes on the meiosis I spindle remain poorly understood. In budding yeast meiosis I, Mad2 and Mad3BUBR1 are equally important for spindle checkpoint delay, but biorientation of homologs on the meiosis I spindle requires Mad2, but not Mad3BUBR1.3,4 Here we reveal the distinct functions of Mad2 and Mad3BUBR1 in meiosis I chromosome segregation. Mad2 promotes the prophase to metaphase I transition, while Mad3BUBR1 associates with the TOGL1 domain of Stu1CLASP, a conserved plus-end microtubule protein that is important for chromosome capture onto the spindle. Homologous chromosome pairs that are proficient in crossover formation but fail to biorient rely on Mad3BUBR1-Stu1CLASP to ensure their efficient attachment to microtubules and segregation during meiosis I. Furthermore, we show that Mad3BUBR1-Stu1CLASP are essential to rescue the segregation of mini-chromosomes lacking crossovers. Our findings define a new pathway ensuring microtubule-dependent chromosome capture and demonstrate that spindle checkpoint proteins safeguard the fidelity of chromosome segregation both by actively promoting chromosome alignment and by delaying cell-cycle progression until this has occurred.

Cytoskeleton-associated protein 2-like (CKAP2L) is a paralogue of cytoskeleton-associated protein 2 (CKAP2). We characterized the expression pattern, subcellular localization, and microtubule-stabilizing properties of human CKAP2L. The levels of both CKAP2L transcript and protein were cell cycle phase-dependent, peaking during the G2/M phase and relatively high in certain human tissues, including testis, intestine, and spleen. CKAP2L protein was detectable in all human cancer cell lines we tested. CKAP2L localized to the mitotic spindle apparatus during mitosis, as reported previously. During interphase, however, CKAP2L localized mainly to the nucleus. Ectopic overexpression of CKAP2L resulted in \'microtubule bundling\', and, consequently, an elevated CKAP2L level led to prolonged mitosis. These findings support the mitotic role of CKAP2L during the human cell cycle.

The primary cilium is a small organelle protruding from the cell surface and receives signals from the extracellular milieu. While dozens of studies have reported that several genetic factors impair the structure of primary cilia, evidence for environmental stimuli affecting primary cilia structures is limited. Here, we investigated an extracellular stress that affected primary cilia morphology and its underlying mechanisms. Hyperosmotic shock induced reversible shortenings and disassembly of primary cilia in murine intramedullary collecting duct cells. The primary cilia shortening caused by hyperosmotic shock followed delocalization of pericentriolar materials (PCMs). Excessive microtubule and F-actin formation in the cytoplasm coincided with those hyperosmotic shock-induced changes of primary cilia and PCMs. A microtubule-disrupting agent, Nocodazole, prevented the hyperosmotic shock-induced primary cilia disassembly partially, while preventing the delocalization of PCMs almost 100%. An actin polymerization inhibitor, Latrunculin A, also prevented partially the hyperosmotic shock-induced primary cilia shortening and disassembly, while preventing the delocalization of PCMs almost 100%. We demonstrate that hyperosmotic shock induces reversible morphological changes in primary cilia and PCMs in a manner dependent on excessive formation of microtubule and F-actin.

Karyotypes, composed of chromosomes, must be accurately partitioned by the mitotic spindle for optimal cell health. However, it is unknown how underlying characteristics of karyotypes, such as chromosome number and size, govern the scaling of the mitotic spindle to ensure accurate chromosome segregation and cell proliferation. We utilize budding yeast strains engineered with fewer chromosomes, including just two \"mega chromosomes,\" to study how spindle size and function are responsive to, and scaled by, karyotype. We determined that deletion and overexpression of spindle-related genes are detrimental to the growth of strains with two chromosomes, suggesting that mega chromosomes exert altered demands on the spindle. Using confocal microscopy, we demonstrate that cells with fewer but longer chromosomes have smaller spindle pole bodies, fewer microtubules, and longer spindles. Moreover, using electron tomography and confocal imaging, we observe elongated, bent anaphase spindles with fewer core microtubules in strains with mega chromosomes. Cells harboring mega chromosomes grow more slowly, are delayed in mitosis, and a subset struggle to complete chromosome segregation. We propose that the karyotype of the cell dictates the microtubule number, type, spindle pole body size, and spindle length, subsequently influencing the dynamics of mitosis, such as the rate of spindle elongation and the velocity of pole separation. Taken together, our results suggest that mitotic spindles are highly plastic ultrastructures that can accommodate and adjust to a variety of karyotypes, even within a species.