Microtubule Shaft Research Articles

Microtubule shaft integrity emerges as a crucial determinant of the acetylation pattern.

Microtubule shaft integrity emerges as a crucial determinant of the acetylation pattern.

Microtubule damage shapes the acetylation gradient

The properties of single microtubules within the microtubule network can be modulated through post-translational modifications (PTMs), including acetylation within the lumen of microtubules. To access the lumen, the enzymes could enter through the microtubule ends and at damage sites along the microtubule shaft. Here we show that the acetylation profile depends on damage sites, which can be caused by the motor protein kinesin-1. Indeed, the entry of the deacetylase HDAC6 into the microtubule lumen can be modulated by kinesin-1-induced damage sites. In contrast, activity of the microtubule acetylase αTAT1 is independent of kinesin-1-caused shaft damage. On a cellular level, our results show that microtubule acetylation distributes in an exponential gradient. This gradient results from tight regulation of microtubule (de)acetylation and scales with the size of the cells. The control of shaft damage represents a mechanism to regulate PTMs inside the microtubule by giving access to the lumen.

The motor domain of the kinesin Kip2 promotes microtubule polymerization at microtubule tips.

Kinesins are microtubule-dependent motor proteins, some of which moonlight as microtubule polymerases, such as the yeast protein Kip2. Here, we show that the CLIP-170 ortholog Bik1 stabilizes Kip2 at microtubule ends where the motor domain of Kip2 promotes microtubule polymerization. Live-cell imaging and mathematical estimation of Kip2 dynamics reveal that disrupting the Kip2-Bik1 interaction aborts Kip2 dwelling at microtubule ends and abrogates its microtubule polymerization activity. Structural modeling and biochemical experiments identify a patch of positively charged residues that enables the motor domain to bind free tubulin dimers alternatively to the microtubule shaft. Neutralizing this patch abolished the ability of Kip2 to promote microtubule growth both in vivo and in vitro without affecting its ability to walk along microtubules. Our studies suggest that Kip2 utilizes Bik1 as a cofactor to track microtubule tips, where its motor domain then recruits free tubulin and catalyzes microtubule assembly.

Theory of tip structure–dependent microtubule catastrophes and damage-induced microtubule rescues

Microtubules are essential cytoskeletal polymers that exhibit stochastic switches between tubulin assembly and disassembly. Here, we examine possible mechanisms for these switches, called catastrophes and rescues. We formulate a four-state Monte Carlo model, explicitly considering two biochemical and two conformational states of tubulin, based on a recently conceived view of microtubule assembly with flared ends. The model predicts that high activation energy barriers for lateral tubulin interactions can cause lagging of curled protofilaments, leading to a ragged appearance of the growing tip. Changes in the extent of tip raggedness explain some important but poorly understood features of microtubule catastrophe: weak dependence on tubulin concentration and an increase in its probability over time, known as aging. The model predicts a vanishingly rare frequency of spontaneous rescue unless patches of guanosine triphosphate tubulin are artificially embedded into microtubule lattice. To test our model, we used invitro reconstitution, designed to minimize artifacts induced by microtubule interaction with nearby surfaces. Microtubules were assembled from seeds overhanging from microfabricated pedestals and thus well separated from the coverslip. This geometry reduced the rescue frequency and the incorporation of tubulins into the microtubule shaft compared with the conventional assay, producing data consistent with the model. Moreover, the rescue positions of microtubules nucleated from coverslip-immobilized seeds displayed a nonexponential distribution, confirming that coverslips can affect microtubule dynamics. Overall, our study establishes a unified theory accounting for microtubule assembly with flared ends, a tip structure-dependent catastrophe frequency, and a microtubule rescue frequency dependent on lattice damage and repair.

Mechanisms of Motor-Independent Membrane Remodeling Driven by Dynamic Microtubules.

SummaryMicrotubule-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.

Lattice defects induce microtubule self-renewal.

Microtubules are dynamic polymers, which grow and shrink by addition and removal of tubulin dimers at their extremities. Within the microtubule shaft, dimers adopt a densely packed and highly ordered crystal-like lattice structure, which is generally not considered to be dynamic. Here we report that thermal forces are sufficient to remodel the microtubule shaft, despite its apparent stability. Our combined experimental data and numerical simulations on lattice dynamics and structure suggest that dimers can spontaneously leave and be incorporated into the lattice at structural defects. We propose a model mechanism, where the lattice dynamics is initiated via a passive breathing mechanism at dislocations, which are frequent in rapidly growing microtubules. These results show that we may need to extend the concept of dissipative dynamics, previously established for microtubule extremities, to the entire shaft, instead of considering it as a passive material.

Severing Enzymes Amplify Microtubule Arrays through Lattice GTP-Tubulin Incorporation

Spastin and katanin sever and destabilize microtubules. Their mutation causes debilitating diseases. Paradoxically, despite their destructive activity they increase microtubule mass in spindles, plant cortical arrays and neurons. Combining electron and single-molecule TIRF microscopy we show that the elemental step in microtubule-severing is the generation of nanoscale damage throughout the microtubule by active extraction of tubulin heterodimers. These damage sites are repaired spontaneously by GTP-tubulin incorporation. As a result, the microtubule shaft is rejuvenated with GTP-tubulin islands that stabilize it against depolymerization and newly severed ends emerge with a high-density of GTP-tubulin that protects against depolymerization. Consequently, spastin and katanin increase microtubule rescue frequency without affecting growth rates and catastrophe. The stabilization of the newly severed plus-ends and the higher rescue frequency synergize to amplify microtubule number and mass. Thus, severing enzymes regulate microtubule architecture and dynamics by promoting GTP-tubulin incorporation within the microtubule shaft.

Strengthening Microtubules by Cuts that Heal

Microtubule-severing enzymes, which can remove tubulin dimers from microtubule lattices, participate in cytoskeletal remodeling in various contexts. A recent study showed that partially damaged microtubule shafts and new microtubule ends generated by these enzymes can incorporate GTP-tubulin and serve as sites of microtubule rescue and re-growth, explaining how severing enzymes can amplify microtubule arrays.

Severing to build microtubules

Cytoskeleton![Figure][1] New tubulin (pink) patches small holes in microtubule lattices made by severing enzymes (purple). CREDIT: JANET IWASA Microtubules are essential intracellular polymers, built from tubulin subunits, that establish cell shape, move organelles, and segregate chromosomes during cell division. Vemu et al. show that microtubule-severing enzymes extract tubulin subunits along the microtubule shaft. This nanoscale damage is repaired by the incorporation of free tubulin, which stabilizes the microtubule against depolymerization. When extraction outpaces repair, microtubules are severed, emerging with stabilized ends composed of fresh tubulin. The severed microtubules act as templates for new microtubule growth, leading to amplification of microtubule number and mass. Thus, seemingly paradoxically, severing enzymes can increase microtubule mass in processes such as neurogenesis and mitotic spindle assembly. Science , this issue p. [eaau1504][2] [1]: pending:yes [2]: /lookup/doi/10.1126/science.aau1504

Severing enzymes amplify microtubule arrays through lattice GTP-tubulin incorporation.

Spastin and katanin sever and destabilize microtubules. Paradoxically, despite their destructive activity they increase microtubule mass in vivo. We combined single-molecule total internal reflection fluorescence microscopy and electron microscopy to show that the elemental step in microtubule severing is the generation of nanoscale damage throughout the microtubule by active extraction of tubulin heterodimers. These damage sites are repaired spontaneously by guanosine triphosphate (GTP)-tubulin incorporation, which rejuvenates and stabilizes the microtubule shaft. Consequently, spastin and katanin increase microtubule rescue rates. Furthermore, newly severed ends emerge with a high density of GTP-tubulin that protects them against depolymerization. The stabilization of the newly severed plus ends and the higher rescue frequency synergize to amplify microtubule number and mass. Thus, severing enzymes regulate microtubule architecture and dynamics by promoting GTP-tubulin incorporation within the microtubule shaft.

Spectraplakins Promote Microtubule-Mediated Axonal Growth by Functioning As Structural Microtubule-Associated Proteins and EB1-Dependent +TIPs (Tip Interacting Proteins)

The correct outgrowth of axons is essential for the development and regeneration of nervous systems. Axon growth is primarily driven by microtubules. Key regulators of microtubules in this context are the spectraplakins, a family of evolutionarily conserved actin-microtubule linkers. Loss of function of the mouse spectraplakin ACF7 or of its close Drosophila homolog Short stop/Shot similarly cause severe axon shortening and microtubule disorganization. How spectraplakins perform these functions is not known. Here we show that axonal growth-promoting roles of Shot require interaction with EB1 (End binding protein) at polymerizing plus ends of microtubules. We show that binding of Shot to EB1 requires SxIP motifs in Shot's C-terminal tail (Ctail), mutations of these motifs abolish Shot functions in axonal growth, loss of EB1 function phenocopies Shot loss, and genetic interaction studies reveal strong functional links between Shot and EB1 in axonal growth and microtubule organization. In addition, we report that Shot localizes along microtubule shafts and stabilizes them against pharmacologically induced depolymerization. This function is EB1-independent but requires net positive charges within Ctail which essentially contribute to the microtubule shaft association of Shot. Therefore, spectraplakins are true members of two important classes of neuronal microtubule regulating proteins: +TIPs (tip interacting proteins; plus end regulators) and structural MAPs (microtubule-associated proteins). From our data we deduce a model that relates the different features of the spectraplakin C terminus to the two functions of Shot during axonal growth.

Tau pathology: predictive diagnostics, targeted preventive and personalized medicine and application of advanced research in medical practice

Microtubules are key cytoskeletal elements found in all eukaryotic cells. The microtubule shaft is composed of the heterodimer protein, tubulin and decorated with multiple microtubule associated protein, regulating microtubule function. Tau (tubulin associated unit) or MAPT (microtubule associated protein tau), among the first microtubule associated proteins to be identified, was implicated in microtubule initiation as well as assembly, with increased expression in neurons and specific association with axonal microtubules. Alzheimer’s disease (AD) is the most prevalent tauopathy, exhibiting tau-neurofibrillary tangles associated with cognitive dysfunction. AD is also characterized by β-amyloid plaques. An abundance of tau inclusions, in the absence of β-amyloid deposits, can be found in Pick’s disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and other diseases, collectively described as tauopathies. The increase in tau pathology in AD correlates with the associated cognitive decline. The current manuscript touches on the variability as well as common denominators of the various tau pathologies coupled to new approaches/current innovation in treatment of tauopathies in favor of advanced technologies in predictive diagnostics, targeted preventive and personalized medicine (PPPM).

Hematopoietic-specific β1 tubulin participates in a pathway of platelet biogenesis dependent on the transcription factor NF-E2

AbstractThe cellular and molecular bases of platelet release by terminally differentiated megakaryocytes represent important questions in cell biology and hematopoiesis. Mice lacking the transcription factor NF-E2 show profound thrombocytopenia, and their megakaryocytes fail to produce proplatelets, the microtubule-based precursors of blood platelets. Using mRNA subtraction between normal and NF-E2–deficient megakaryocytes, cDNA was isolated encoding β1 tubulin, the most divergent β tubulin isoform. In NF-E2–deficient megakaryocytes, β1 tubulin mRNA and protein are virtually absent. The expression of β1 tubulin is exquisitely restricted to platelets and megakaryocytes, where it appears late in differentiation and localizes to microtubule shafts and coils within proplatelets. Restoring NF-E2 activity in a megakaryoblastic cell line or in NF-E2–deficient primary megakaryocytes rescues the expression of β1 tubulin. Re-expressing β1 tubulin in isolation does not, however, restore proplatelet formation in the defective megakaryocytes, indicating that other critical factors are required; indeed, other genes identified by mRNA subtraction also encode structural and regulatory components of the cytoskeleton. These findings provide critical mechanistic links between NF-E2, platelet formation, and selected microtubule proteins, and they also provide novel molecular insights into thrombopoiesis.

Hematopoietic-specific β1 tubulin participates in a pathway of platelet biogenesis dependent on the transcription factor NF-E2

The cellular and molecular bases of platelet release by terminally differentiated megakaryocytes represent important questions in cell biology and hematopoiesis. Mice lacking the transcription factor NF-E2 show profound thrombocytopenia, and their megakaryocytes fail to produce proplatelets, the microtubule-based precursors of blood platelets. Using mRNA subtraction between normal and NF-E2–deficient megakaryocytes, cDNA was isolated encoding β1 tubulin, the most divergent β tubulin isoform. In NF-E2–deficient megakaryocytes, β1 tubulin mRNA and protein are virtually absent. The expression of β1 tubulin is exquisitely restricted to platelets and megakaryocytes, where it appears late in differentiation and localizes to microtubule shafts and coils within proplatelets. Restoring NF-E2 activity in a megakaryoblastic cell line or in NF-E2–deficient primary megakaryocytes rescues the expression of β1 tubulin. Re-expressing β1 tubulin in isolation does not, however, restore proplatelet formation in the defective megakaryocytes, indicating that other critical factors are required; indeed, other genes identified by mRNA subtraction also encode structural and regulatory components of the cytoskeleton. These findings provide critical mechanistic links between NF-E2, platelet formation, and selected microtubule proteins, and they also provide novel molecular insights into thrombopoiesis.