Opto-Katanin: An Optogenetic Tool for Localized Microtubule Disassembly

Microtubules are major cytoskeletal filaments that drive chromosome separation during cell division, serve as rails for intracellular transport and as a scaffold for organelle positioning. Experimental manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubule integrity are currently lacking. Here, we exploit the dependence of the mammalian microtubule-severing protein katanin on microtubule-targeting co-factors to generate a light-activated system for localized microtubule disassembly that we named opto-katanin. Targeted illumination with blue light induces rapid and localized opto-katanin recruitment and local microtubule depolymerization, which is quickly reversible after stopping light-induced activation. Opto-katanin can be employed to locally perturb microtubule-based transport and organelle morphology in dividing cells and differentiated neurons with high spatiotemporal precision. We show that different microtubule-associated proteins can be used to recruit opto-katanin to microtubules and induce severing, paving the way for spatiotemporally precise manipulation of specific microtubule subpopulations.

The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin

Global regulation of spindle-associated proteins is crucial in oocytes due to the absence of centrosomes and their very large cytoplasmic volume, but little is known about how this is achieved beyond involvement of the Ran-importin pathway. We previously uncovered a novel regulatory mechanism in Drosophila oocytes, in which the phospho-docking protein 14-3-3 suppresses microtubule binding of Kinesin-14/Ncd away from chromosomes. Here we report systematic identification of microtubule-associated proteins regulated by 14-3-3 from Drosophila oocytes. Proteins from ovary extract were co-sedimented with microtubules in the presence or absence of a 14-3-3 inhibitor. Through quantitative mass-spectrometry, we identified proteins or complexes whose ability to binding microtubules is suppressed by 14-3-3, including the chromosomal passenger complex (CPC), the centralspindlin complex and Kinesin-14/Ncd. We showed that 14-3-3 binds to the disordered region of Borealin, and this binding is regulated differentially by two phosphorylations on Borealin. Mutations at these two phospho-sites compromised normal Borealin localisation and centromere bi-orientation in oocytes, showing that phospho-regulation of 14-3-3 binding is important for Borealin localisation and function. The mass spectrometry data are available from ProteomeXchange, identifier ID to be provided when available, PXD000xxx.

The Reduced Longitudinal Growth Induced by Overexpression of pPLAIIIγ Is Regulated by Genes Encoding Microtubule-Associated Proteins

There are three subfamilies of patatin-related phospholipase A (pPLA) group of genes: pPLAI, pPLAII, and pPLAIII. Among the four members of pPLAIIIs (α, β, γ, δ), the overexpression of three isoforms (α, β, and δ) displayed distinct morphological growth patterns, in which the anisotropic cell expansion was disrupted. Here, the least studied pPLAIIIγ was characterized, and it was found that the overexpression of pPLAIIIγ in Arabidopsis resulted in longitudinally reduced cell expansion patterns, which are consistent with the general phenotype induced by pPLAIIIs overexpression. The microtubule-associated protein MAP18 was found to be enriched in a pPLAIIIδ overexpressing line in a previous study. This indicates that factors, such as microtubules and ethylene biosynthesis, are involved in determining the radial cell expansion patterns. Microtubules have long been recognized to possess functional key roles in the processes of plant cells, including cell division, growth, and development, whereas ethylene treatment was reported to induce the reorientation of microtubules. Thus, the possible links between the altered anisotropic cell expansion and microtubules were studied. Our analysis revealed changes in the transcriptional levels of microtubule-associated genes, as well as phospholipase D (PLD) genes, upon the overexpression of pPLAIIIγ. Overall, our results suggest that the longitudinally reduced cell expansion observed in pPLAIIIγ overexpression is driven by microtubules via transcriptional modulation of the PLD and MAP genes. The altered transcripts of the genes involved in ethylene-biosynthesis in pPLAIIIγOE further support the conclusion that the typical phenotype is derived from the link with microtubules.

Tau in Health and Neurodegenerative Diseases

Tau, one of the major microtubule-associated proteins, modulates the dynamic properties of microtubules in the mammalian nervous system. Tau is abundantly expressed in the brain, particularly in the hippocampus. Insoluble and filamentous inclusions of tau in neurons or glia are discovered in neurodegenerative diseases termed ‘tauopathies’, including Alzheimer’s disease (AD), argyrophilic grain disease (AGD), corticobasal degeneration (CBD), frontotemporal dementia (FTD), Pick’s disease (PiD) and progressive supranuclear palsy (PSP). Accumulation of intracellular neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated tau, is directly correlated with the degree of Alzheimer\'s dementia. This chapter reviews the role of tau protein in physiological conditions and the pathological changes of tau related to neurodegenerative diseases. The applications of tau as a therapeutic target are also discussed.

Microtubule-associated proteins promote microtubule generation in the absence of γ-tubulin in human colon cancer cells

The γ-tubulin complex acts as the predominant microtubule (MT) nucleator that initiates MT formation and is therefore an essential factor for cell proliferation. Nonetheless, cellular MTs are formed after experimental depletion of the γ-tubulin complex, suggesting that cells possess other factors that drive MT nucleation. Here, by combining gene knockout, auxin-inducible degron, RNA interference, MT depolymerization/regrowth assay, and live microscopy, we identified four microtubule-associated proteins (MAPs), ch-TOG, CLASP1, CAMSAPs, and TPX2, which are involved in γ-tubulin–independent MT generation in human colon cancer cells. In the mitotic MT regrowth assay, nucleated MTs organized noncentriolar MT organizing centers (ncMTOCs) in the absence of γ-tubulin. Depletion of CLASP1 or TPX2 substantially delayed ncMTOC formation, suggesting that these proteins might promote MT nucleation in the absence of γ-tubulin. In contrast, depletion of ch-TOG or CAMSAPs did not affect the timing of ncMTOC appearance. CLASP1 also accelerates γ-tubulin–independent MT regrowth during interphase. Thus, MT generation can be promoted by MAPs without the γ-tubulin template.

Cardiomyocyte Microtubules: Control of Mechanics, Transport, and Remodeling

Microtubules are essential cytoskeletal elements found in all eukaryotic cells. The structure and composition of microtubules regulate their function, and the dynamic remodeling of the network by posttranslational modifications and microtubule-associated proteins generates diverse populations of microtubules adapted for various contexts. In the cardiomyocyte, the microtubules must accommodate the unique challenges faced by a highly contractile, rigidly structured, and long-lasting cell. Through their canonical trafficking role and positioning of mRNA, proteins, and organelles, microtubules regulate essential cardiomyocyte functions such as electrical activity, calcium handling, protein translation, and growth. In a more specialized role, posttranslationally modified microtubules form load-bearing structures that regulate myocyte mechanics and mechanotransduction. Modified microtubules proliferate in cardiovascular diseases, creating stabilized resistive elements that impede cardiomyocyte contractility and contribute to contractile dysfunction. In this review, we highlight the most exciting new concepts emerging from recent studies into canonical and noncanonical roles of cardiomyocyte microtubules. Expected final online publication date for the Annual Review of Physiology, Volume 84 is February 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Expression of Microtubule-Associated Proteins in Relation to Prognosis and Efficacy of Immunotherapy in Non-Small Cell Lung Cancer

BackgroundMicrotubule-associated proteins (MAPs) have been considered to play significant roles in the tumor evolution of non-small cell lung cancer (NSCLC). Nevertheless, mRNA transcription levels and prognostic value of distinct MAPs in patients with NSCLC remain to be clarified.MethodsIn this study, the Oncomine database, Gene Expression Profiling Interactive Analysis (GEPIA) database, and Human Protein Atlas were utilized to analyze the relationship between mRNA/protein expression of different MAPs and clinical characteristics in NSCLC patients, including tumor type and pathological stage. The correlation between the transcription level of MAPs and overall survival (OS) of NSCLC patients was analyzed by Kaplan–Meier plotter. Besides, 50 frequently altered neighbor genes of the MAPs were screened out, and a network has been constructed via the cBioPortal and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) dataset. Meanwhile, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on the expression data of MAPs and their 50 frequently altered neighbor genes in NSCLC tissues. Furthermore, The Cancer Immunome Atlas (TCIA) was utilized to analyze the relationship between MAP expression and the response to immunotherapy. Finally, we used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to verify the expression of MAPs in 20 patients with NSCLC.ResultsThe present study discovered that the mRNA transcription levels of MAP7/7D2 were enriched in NSCLC tissues, while those of the MAP2/4/6/7D3 were lower in NSCLC specimens than those in control specimens. The mRNA transcription level of MAP6 was significantly associated with the advanced stage of NSCLC. Besides, survival analysis indicated that higher mRNA expressions of MAP2/4/6/7/7D3 were correlated considerably with favorable OS of NSCLC patients, whereas increased mRNA expression levels of MAP1A/1S were associated with poor OS. Moreover, the expression of MAP1A/1B/1S/4/6/7D1/7D3 was significantly correlated with immunophenoscore (IPS) in NSCLC patients.ConclusionsOur analysis indicated that MAP1A/1S could serve as potential personalized therapeutic targets for patients with NSCLC, and the enriched MAP2/4/6/7/7D3 expression could serve as a biomarker for favorable prognosis in NSCLC. Besides, the expression of MAP1A/1B/1S/4/6/7D1/7D3 was closely related to the response to immunotherapy. Taken together, MAP expression has potential application value in the clinical treatment and prognosis assessment of NSCLC patients, and further verifiable experiments can be conducted to verify our results.

Conjugation of Aminoadamantane and γ-Carboline Pharmacophores Gives Rise to Unexpected Properties of Multifunctional Ligands

A new series of conjugates of aminoadamantane and γ-carboline, which are basic scaffolds of the known neuroactive agents, memantine and dimebon (Latrepirdine) was synthesized and characterized. Conjugates act simultaneously on several biological structures and processes involved in the pathogenesis of Alzheimer’s disease and some other neurodegenerative disorders. In particular, these compounds inhibit enzymes of the cholinesterase family, exhibiting higher inhibitory activity against butyrylcholinesterase (BChE), but having almost no effect on the activity of carboxylesterase (anti-target). The compounds serve as NMDA-subtype glutamate receptor ligands, show mitoprotective properties by preventing opening of the mitochondrial permeability transition (MPT) pore, and act as microtubule stabilizers, stimulating the polymerization of tubulin and microtubule-associated proteins. Structure–activity relationships were studied, with particular attention to the effect of the spacer on biological activity. The synthesized conjugates showed new properties compared to their prototypes (memantine and dimebon), including the ability to bind to the ifenprodil-binding site of the NMDA receptor and to occupy the peripheral anionic site of acetylcholinesterase (AChE), which indicates that these compounds can act as blockers of AChE-induced β-amyloid aggregation. These new attributes of the conjugates represent improvements to the pharmacological profiles of the separate components by conferring the potential to act as neuroprotectants and cognition enhancers with a multifunctional mode of action.

Identification of disease-relevant modulators of the SHH pathway in the developing brain

ABSTRACT Pathogenic gene variants in humans that affect the sonic hedgehog (SHH) pathway lead to severe brain malformations with variable penetrance due to unknown modifier genes. To identify such modifiers, we established novel congenic mouse models. LRP2-deficient C57BL/6N mice suffer from heart outflow tract defects and holoprosencephaly caused by impaired SHH activity. These defects are fully rescued on a FVB/N background, indicating a strong influence of modifier genes. Applying comparative transcriptomics, we identified Pttg1 and Ulk4 as candidate modifiers upregulated in the rescue strain. Functional analyses showed that ULK4 and PTTG1, both microtubule-associated proteins, are positive regulators of SHH signaling, rendering the pathway more resilient to disturbances. In addition, we characterized ULK4 and PTTG1 as previously unidentified components of primary cilia in the neuroepithelium. The identification of genes that powerfully modulate the penetrance of genetic disturbances affecting the brain and heart is likely relevant to understanding the variability in human congenital disorders.