Microtubule Organization In Cells Research Articles

CLASP-mediated competitive binding in protein condensates directs microtubule growth

Microtubule organization in cells relies on targeting mechanisms. Cytoplasmic linker proteins (CLIPs) and CLIP-associated proteins (CLASPs) are key regulators of microtubule organization, yet the underlying mechanisms remain elusive. Here, we reveal that the C-terminal domain of CLASP2 interacts with a common motif found in several CLASP-binding proteins. This interaction drives the dynamic localization of CLASP2 to distinct cellular compartments, where CLASP2 accumulates in protein condensates at the cell cortex or the microtubule plus end. These condensates physically contact each other via CLASP2-mediated competitive binding, determining cortical microtubule targeting. The phosphorylation of CLASP2 modulates the dynamics of the condensate-condensate interaction and spatiotemporally navigates microtubule growth. Moreover, we identify additional CLASP-interacting proteins that are involved in condensate contacts in a CLASP2-dependent manner, uncovering a general mechanism governing microtubule targeting. Our findings not only unveil a tunable multiphase system regulating microtubule organization, but also offer general mechanistic insights into intricate protein-protein interactions at the mesoscale level.

Single-motor and multi-motor motility properties of kinesin-6 family members.

ABSTRACTKinesin motor proteins are responsible for orchestrating a variety of microtubule-based processes including intracellular transport, cell division, cytoskeletal organization, and cilium function. Members of the kinesin-6 family play critical roles in anaphase and cytokinesis during cell division as well as in cargo transport and microtubule organization during interphase, however little is known about their motility properties. We find that truncated versions of MKLP1 (HsKIF23), MKLP2 (HsKIF20A), and HsKIF20B largely interact statically with microtubules as single molecules but can also undergo slow, processive motility, most prominently for MKLP2. In multi-motor assays, all kinesin-6 proteins were able to drive microtubule gliding and MKLP1 and KIF20B were also able to drive robust transport of both peroxisomes, a low-load cargo, and Golgi, a high-load cargo, in cells. In contrast, MKLP2 showed minimal transport of peroxisomes and was unable to drive Golgi dispersion. These results indicate that the three mammalian kinesin-6 motor proteins can undergo processive motility but differ in their ability to generate forces needed to drive cargo transport and microtubule organization in cells.

A pathogenic tau fragment compromises microtubules, disrupts insulin signaling and induces the unfolded protein response

Human tauopathies including Alzheimer’s disease, progressive supranuclear palsy and related disorders, are characterized by deposition of pathological forms of tau, synaptic dysfunction and neuronal loss. We have previously identified a pathogenic C-terminal tau fragment (Tau35) that is associated with human tauopathy. However, it is not known how tau fragmentation affects critical molecular processes in cells and contributes to impaired physiological function. Chinese hamster ovary (CHO) cells and new CHO cell lines stably expressing Tau35 or full-length human tau were used to compare the effects of disease-associated tau cleavage on tau function and signaling pathways. Western blots, microtubule-binding assays and immunofluorescence labeling were used to examine the effects of Tau35 on tau function and on signaling pathways in CHO cells. We show that Tau35 undergoes aberrant phosphorylation when expressed in cells. Although Tau35 contain the entire microtubule-binding region, the lack of the amino terminal half of tau results in a marked reduction in microtubule binding and defective microtubule organization in cells. Notably, Tau35 attenuates insulin-mediated activation of Akt and a selective inhibitory phosphorylation of glycogen synthase kinase-3. Moreover, Tau35 activates ribosomal protein S6 kinase beta-1 signaling and the unfolded protein response, leading to insulin resistance in cells. Tau35 has deleterious effects on signaling pathways that mediate pathological changes and insulin resistance, suggesting a mechanism through which N-terminal cleavage of tau leads to the development and progression of tau pathology in human tauopathy. Our findings highlight the importance of the N-terminal region of tau for its normal physiological function. Furthermore, we show that pathogenic tau cleavage induces tau phosphorylation, resulting in impaired microtubule binding, disruption of insulin signaling and activation of the unfolded protein response. Since insulin resistance is a feature of several tauopathies, this work suggests new potential targets for therapeutic intervention.

Katanin Activity is Regulated by Common Cellular Components

Proper organization of the microtubule cytoskeletal network is required to perform necessary cellular functions. Network organization is achieved through remodeling by microtubule associated proteins (MAPs) that control microtubule dynamics by selectively stabilizing and destabilizing microtubules. Stabilizing MAPs are relatively well understood, while less is known about destabilizing MAPs, such as severing enzymes. Katanin, the first-discovered microtubule severing enzyme, is a AAA+ enzyme that oligomerizes into hexamers and uses ATP hydrolysis to sever microtubules. Using single molecule biophysics techniques in vitro we investigate how katanin and its severing activity can be regulated by the type of microtubule, free tubulin, ATP concentration, and the neuronal MAP tau isoform 4RL. This work provides evidence that katanin activity is regulated by common cellular contents that will ultimately affect microtubule organization in cells.

Cytotoxic Effects of Bilberry Extract on MCF7-GFP-Tubulin Breast Cancer Cells

Bilberry (European blueberry) has been reported to have many biological effects, including anticancer activity. In this study, we investigated the antiproliferative effects of bilberry extract in relation to its ability to induce apoptosis and affect microtubule assembly and organization in MCF7 human breast cancer cells. We observed that bilberry extract inhibited cell proliferation in a concentration-dependent fashion with a 50% inhibitory concentration of 0.3-0.4 mg/mL, in concert with induction of apoptotic cell death. At these concentrations there was no selective inhibition of mitosis or any other cell cycle stage, nor was there any apparent effect on the microtubule or actin cytoskeletons. However, somewhat higher extract concentrations (0.5-0.9 mg/mL) did cause an increase in the fraction of cells at the G(2)/M phase of the cell cycle, together with destruction of microtubules and formation of punctate tubulin aggregates in the cells. Bilberry extract at 0.3-0.4 mg/mL did not appreciably inhibit microtubule polymerization in vitro, but significant inhibition of polymerization (approximately 30%) did occur at higher extract concentrations (0.5-1 mg/mL). We conclude that bilberry extract as ingested by humans, not just the purified anthocyanins it contains, inhibits proliferation of and induces apoptosis in breast cancer cells at its lowest effective concentrations via a mechanism that does not involve action on microtubules or on mitosis. We further conclude that at somewhat higher concentrations the extract modifies microtubule organization in cells and causes accumulation of cells at mitosis by a direct action on microtubules.

Effects of anti-microtubule agents on microtubule organization in cells lacking the kinesin-13 MCAK

Dynamic microtubules are necessary for proper mitotic spindle assembly and chromosome segregation during mitosis. Members of the kinesin superfamily of molecular motor proteins are important to spindle function. Of particular interest is the Kinesin-13 family member MCAK, which acts to regulate microtubule dynamics during spindle assembly and to ensure proper attachments of chromosomes to spindle microtubules. The unique ability of MCAK to regulate microtubule dynamics makes it a potential target for development of new drugs that alter spindle function. Here, we knocked down MCAK via RNAi in normal and malignant cell lines and found that the two tested malignant cell lines were acutely sensitive to MCAK knockdown, while the tested normal cells were less sensitive. In addition, we looked at the effect of combining MCAK knockdown and drug treatment with paclitaxel or vinblastine to identify spindle assembly defects. We found that MCAK knockdown increased the morphological defects of the microtubule cytoskeleton in HeLa cells caused by anti-microtubule drugs. Our studies support the idea that MCAK would be a good target for new chemotherapeutic development and may be particular useful in combination therapies with currently available anti-microtubule agents.

Microtubule transport, concentration and alignment in enclosed microfluidic channels

The kinesin-microtubule system has emerged as a versatile model system for biologically-derived microscale transport. While kinesin motors in cells transport cargo along static microtubule tracks, for in vitro transport applications it is preferable to invert the system and transport cargo-functionalized microtubules along immobilized kinesin motors. However, for efficient cargo transport and to enable this novel transport system to be interfaced with traditional microfluidics, it is important to fabricate enclosed microchannels that are compatible with kinesin motors and microtubules, that enable fluorescence imaging of microtubule movement, and that provide fluidic connections for sample introduction. Here we construct a three-tier hierarchical system of microfluidic channels that links microscale transport channels to macroscopic fluid connections. Shallow microchannels (5 microm wide and 1 microm deep) are etched in a glass substrate and bonded to a cover glass using PMMA as an adhesive, while intermediate channels (approximately 100 microm wide) serve as reservoirs and connect to 250 microm deep microchannels that hold fine gauge tubing for fluid injection. To demonstrate the utility of this device, we first show the performance of a directional rectifier that redirects 96% of moving microtubules and, because any microtubules that detach rapidly rebind to the motor-coated surface, suffers no microtubule loss over time. Second, we develop an approach, using a headless kinesin construct, to eliminate gradients in motor adsorption and microtubule binding in the enclosed channels, which enables precise control of kinesin density in the microchannels. Finally, we show that a 60 microm diameter circular ring functionalized with motors concentrates and aligns bundles of approximately 3000 uniformly oriented microtubules, while suffering negligible ATP depletion. These aligned isopolar microtubules are an important tool for microscale transport applications and can be employed as a model in vitro system for studying kinesin-driven microtubule organization in cells.

LIM Kinase 1 Coordinates Microtubule Stability and Actin Polymerization in Human Endothelial Cells

Microtubule (MT) destabilization promotes the formation of actin stress fibers and enhances the contractility of cells; however, the mechanism involved in the coordinated regulation of MTs and the actin cytoskeleton is poorly understood. LIM kinase 1 (LIMK1) regulates actin polymerization by phosphorylating the actin depolymerization factor, cofilin. Here we report that LIMK1 is also involved in the MT destabilization. In endothelial cells endogenous LIMK1 co-localizes with MTs and forms a complex with tubulin via the PDZ domain. MT destabilization induced by thrombin or nocodazole resulted in a decrease of LIMK1 colocalization with MTs. Overexpression of wild type LIMK1 resulted in MT destabilization, whereas the kinase-dead mutant of LIMK1 (KD) did not affect MT stability. Importantly, down-regulation of endogenous LIMK1 by small interference RNA resulted in abrogation of the thrombin-induced MTs destabilization and the inhibition of thrombin-induced actin polymerization. Expression of Rho kinase 2, which phosphorylates and activates LIMK1, dramatically decreases the interaction of LIMK1 with tubulin but increases its interaction with actin. Interestingly, expression of KD-LIMK1 or small interference RNA-LIMK1 prevents thrombin-induced microtubule destabilization and F-actin formation, suggesting that LIMK1 activity is required for thrombin-induced modulation of microtubule destabilization and actin polymerization. Our findings indicate that LIMK1 may coordinate microtubules and actin cytoskeleton.

The effects of glutaraldehyde on the bound nucleotide content and morphology of microtubules in vitro

Guanine nucleotide bound to the exchangeable site on tubulin is known to become nonexchangeable when the tubulin molecule is incorporated into a microtubule. We have found that glutaraldehyde, at concentrations commonly used to fix microtubules for electron microscopy (0.5–2%), causes the rapid loss of bound nucleotide from microtubules. Lower concentrations which still fix microtubules do not cause the release. Examination of negative stained samples with the electron microscope shows an apparent difference in the surface morphology of microtubules fixed with the different concentrations of glutaraldehyde. Thus, glutaraldehyde fixation can produce both structural and biochemical alterations of microtubules, which may be significant in the interpretation of the structural organization of microtubules in cells, especially in terms of the noncovalent association of microtubules with other cytoskeletal components.