Cold Stability Of Microtubules Research Articles

IPSCs from a Hibernator Provide a Platform for Studying Cold Adaptation and Its Potential Medical Applications

Hibernating mammals survive hypothermia (<10°C) without injury, a remarkable feat of cellular preservation that bears significance for potential medical applications. However, mechanisms imparting cold resistance, such as cytoskeleton stability, remain elusive. Using the first iPSC line from a hibernating mammal (13-lined ground squirrel), we uncovered cellular pathways critical for cold tolerance. Comparison between human and ground squirrel iPSC-derived neurons revealed differential mitochondrial and protein quality control responses to cold. In human iPSC-neurons, cold triggered mitochondrial stress, resulting in reactive oxygen species overproduction and lysosomal membrane permeabilization, contributing to microtubule destruction. Manipulations of these pathways endowed microtubule cold stability upon human iPSC-neurons and rat (a non-hibernator)retina, preserving its light responsiveness after prolonged cold exposure. Furthermore, these treatments significantly improved microtubule integrity in cold-stored kidneys, demonstrating the potential for prolonging shelf-life of organ transplants. Thus, ground squirrel iPSCs offer a unique platform for bringing cold-adaptive strategies from hibernators to humans in clinical applications. VIDEO ABSTRACT.

Regulation of cambial activity in relation to environmental conditions: understanding the role of temperature in wood formation of trees

The timing of cambial reactivation plays an important role in determination of the amount and quality of wood and the environmental adaptivity of trees. Environmental factors, such as temperature, influence the growth and development of trees. Temperatures from late winter to early spring affect the physiological processes that are involved in the initiation of cambial cell division and xylem differentiation in trees. Cumulative elevated temperatures from late winter to early spring result in earlier initiation of cambial reactivation and xylem differentiation in tree stems and an extended growth period. However, earlier cambial reactivation increases the risk for frost damage because the cold tolerance of cambium decreases after cambial reactivation. The present review focuses on temperature regulation on the timing of cambial reactivation and xylem differentiation in trees, and also highlights recent advances in our understanding of seasonal changes in the cold stability of microtubules in trees. The review also summarizes the present understanding of the relationships between the timing of cambial reactivation, the start of xylem differentiation and changes in levels of storage materials in trees, as well as an attempt to identify the source of energy for cell division and differentiation. A better understanding of the mechanisms that regulate wood formation in trees and the influence of environmental conditions on such mechanisms should help in efforts to improve and enhance the exploitation of wood for commercial applications and to prepare for climatic change.

Bmcc1s, a Novel Brain-Isoform of Bmcc1, Affects Cell Morphology by Regulating MAP6/STOP Functions

The BCH (BNIP2 and Cdc42GAP Homology) domain-containing protein Bmcc1/Prune2 is highly enriched in the brain and is involved in the regulation of cytoskeleton dynamics and cell survival. However, the molecular mechanisms accounting for these functions are poorly defined. Here, we have identified Bmcc1s, a novel isoform of Bmcc1 predominantly expressed in the mouse brain. In primary cultures of astrocytes and neurons, Bmcc1s localized on intermediate filaments and microtubules and interacted directly with MAP6/STOP, a microtubule-binding protein responsible for microtubule cold stability. Bmcc1s overexpression inhibited MAP6-induced microtubule cold stability by displacing MAP6 away from microtubules. It also resulted in the formation of membrane protrusions for which MAP6 was a necessary cofactor of Bmcc1s. This study identifies Bmcc1s as a new MAP6 interacting protein able to modulate MAP6-induced microtubule cold stability. Moreover, it illustrates a novel mechanism by which Bmcc1 regulates cell morphology.

Structural thermal adaptation of β‐tubulins from the Antarctic psychrophilic protozoan Euplotes focardii

Tubulin dimers of psychrophilic eukaryotes can polymerize into microtubules at 4°C, a temperature at which microtubules from mesophiles disassemble. This unique capability requires changes in the primary structure and/or in post-translational modifications of the tubulin subunits. To contribute to the understanding of mechanisms responsible for microtubule cold stability, here we present a computational structural analysis based on molecular dynamics (MD) and experimental data of three β-tubulin isotypes, named EFBT2, EFBT3, and EFBT4, from the Antarctic protozoon Euplotes focardii that optimal temperature for growth and reproduction is 4°C. In comparison to the β-tubulin from E. crassus, a mesophilic Euplotes species, EFBT2, EFBT3, and EFBT4 possess unique amino acid substitutions that confer different flexible properties of the polypeptide, as well as an increased hydrophobicity of the regions involved in microtubule interdimeric contacts that may overcome the microtubule destabilizing effect of cold temperatures. The structural analysis based on MD indicated that all isotypes display different flexibility properties in the regions involved in the formation of longitudinal and lateral contacts during microtubule polymerization. We also investigated the role of E. focardii β-tubulin isotypes during the process of cilia formation. The unique characteristics of the primary and tertiary structures of psychrophilic β-tubulin isotypes seem responsible for the formation of microtubules with distinct dynamic and functional properties.

Cold stability of microtubules in wood-forming tissues of conifers during seasons of active and dormant cambium

The cold stability of microtubules during seasons of active and dormant cambium was analyzed in the conifers Abies firma, Abies sachalinensis and Larix leptolepis by immunofluorescence microscopy. Samples were fixed at room temperature and at a low temperature of 2-3°C to examine the effects of low temperature on the stability of microtubules. Microtubules were visible in cambium, xylem cells and phloem cells after fixation at room temperature during seasons of active and dormant cambium. By contrast, fixation at low temperature depolymerized microtubules in cambial cells, differentiating tracheids, differentiating xylem ray parenchyma and phloem ray parenchyma cells during the active season. However, similar fixation did not depolymerize microtubules during cambial dormancy in winter. Our results indicate that the stability of microtubules in cambial cells and cambial derivatives at low temperature differs between seasons of active and dormant cambium. Moreover, the change in the stability of microtubules that we observed at low temperature might be closely related to seasonal changes in the cold tolerance of conifers. In addition, low-temperature fixation depolymerized microtubules in cambial cells and differentiating cells that had thin primary cell walls, while such low-temperature fixation did not depolymerize microtubules in differentiating secondary xylem ray parenchyma cells and tracheids that had thick secondary cell walls. The stability of microtubules at low temperature appears to depend on the structure of the cell wall, namely, primary or secondary. Therefore, we propose that the secondary cell wall might be responsible for the cold stability of microtubules in differentiating secondary xylem cells of conifers.

Regulation of Microtubule Assembly and Stability by the Transactivator of Transcription Protein of Jembrana Disease Virus

Microtubules are cytoskeletal polymers consisting of tubulin subunits that take part in diverse cell activities. Many viruses hijack cellular motor proteins to move on microtubules toward the cell interior during the entry process and toward the plasma membrane during the egress period. In addition, viruses often remodel microtubules to facilitate the generation of infectious progeny. In this study, we found that the transactivator of transcription protein of Jembrana disease virus (Jtat) bound tubulin and microtubules both in cells and in the purified system. Microtubule co-sedimentation and co-localization assays revealed a robust interaction of Jtat with microtubules. Tubulin turbidity assay further showed that Jtat promoted tubulin polymerization in vitro in a concentration-dependent manner. Moreover, Jtat promoted the partitioning of cellular tubulin toward the polymeric form, increased the level of tubulin acetylation, and significantly enhanced the cold stability of cellular microtubules. In addition, Jtat-mediated disruption of microtubule dynamics induced the release of Bim from microtubules, leading to profound apoptosis. These results not only identify Jtat as an important viral regulator of microtubule dynamics but also indicate that Jtat-induced apoptosis might contribute to Jembrana disease pathogenesis.

Phosphorylation of Microtubule-associated Protein STOP by Calmodulin Kinase II

STOP proteins are microtubule-associated, calmodulin-regulated proteins responsible for the high degree of stabilization displayed by neuronal microtubules. STOP suppression in mice induces synaptic defects affecting both short and long term synaptic plasticity in hippocampal neurons. Interestingly, STOP has been identified as a component of synaptic structures in neurons, despite the absence of microtubules in nerve terminals, indicating the existence of mechanisms able to induce a translocation of STOP from microtubules to synaptic compartments. Here we have tested STOP phosphorylation as a candidate mechanism for STOP relocalization. We show that, both in vitro and in vivo, STOP is phosphorylated by the multifunctional enzyme calcium/calmodulin-dependent protein kinase II (CaMKII), which is a key enzyme for synaptic plasticity. This phosphorylation occurs on at least two independent sites. Phosphorylated forms of STOP do not bind microtubules in vitro and do not co-localize with microtubules in cultured differentiating neurons. Instead, phosphorylated STOP co-localizes with actin assemblies along neurites or at branching points. Correlatively, we find that STOP binds to actin in vitro. Finally, in differentiated neurons, phosphorylated STOP co-localizes with clusters of synaptic proteins, whereas unphosphorylated STOP does not. Thus, STOP phosphorylation by CaMKII may promote STOP translocation from microtubules to synaptic compartments where it may interact with actin, which could be important for STOP function in synaptic plasticity.

Myelin basic protein functions as a microtubule stabilizing protein in differentiated oligodendrocytes

Myelin basic protein (MBP) is an oligodendrocyte-specific protein essential for oligodendrocyte morphogenesis at late stages of cell differentiation. There is evidence that the morphogenetic function of MBP is mediated by MBP interaction with the cytoskeleton. Thus, an MBP/cytoplasmic microtubule association has been reported, and MBP has Ca(2+)/calmodulin-regulated microtubule cold-stabilizing activity in vitro. However, the unambiguous demonstration of a microtubule-stabilizing activity for MBP in cells has been difficult because oligodendrocytes contain variants of STOP (stable tubule only polypeptide) proteins, which are responsible for microtubule cold stability in different cell types. Herein, we have used genetic mouse models and RNA interference to assay independently the microtubule cold-stabilizing activities of MBP and of STOP in developing oligodendrocytes. In wild-type oligodendrocytes, microtubules were cold stable throughout maturation, which is consistent with the presence of STOP proteins from early stages of differentiation. In contrast, in oligodendrocytes from STOP-deficient mice, microtubules were cold labile in the absence of MBP expression or when MBP expression was restricted to the cell body and became stable in fully differentiated oligodendrocytes, where MBP is expressed in cell extensions. The suppression of MBP by RNA interference in STOP-deficient oligodendrocytes suppressed microtubule cold stability. Additionally, STOP suppression in oligodendrocytes derived from shiverer mice that lack MBP led to the complete suppression of microtubule cold stability at all stages of cell differentiation. These results demonstrate that both STOP and MBP function as microtubule-stabilizing proteins in differentiating oligodendrocytes and could be important for the morphogenetic function of MBP.

Direct Regulation of Microtubule Dynamics by Protein Kinase CK2

Microtubule dynamics is essential for many vital cellular processes such as morphogenesis and motility. Protein kinase CK2 is a ubiquitous protein kinase that is involved in diverse cellular functions. CK2 holoenzyme is composed of two catalytic alpha or alpha' subunits and two regulatory beta subunits. We show that the alpha subunit of CK2 binds directly to both microtubules and tubulin heterodimers. CK2 holoenzyme but neither of its individual subunits exhibited a potent effect of inducing microtubule assembly and bundling. Moreover, the polymerized microtubules were strongly stabilized by CK2 against cold-induced depolymerization. Interestingly, the kinase activity of CK2 is not required for its microtubule-assembling and stabilizing function because a kinase-inactive mutant of CK2 displayed the same microtubule-assembling activity as the wild-type protein. Knockdown of CK2alpha/alpha' in cultured cells by RNA interference dramatically destabilized their microtubule networks, and the destabilized microtubules were readily destructed by colchicine at a very low concentration. Further, over-expression of chicken CK2alpha or its kinaseinactive mutant in the endogenous CK2alpha/alpha'-depleted cells fully restored the microtubule resistance to the low dose of colchicine. Taken together, CK2 is a microtubule-associated protein that confers microtubule stability in a phosphorylation-independent manner.

The suppression of brain cold-stable microtubules in mice induces synaptic defects associated with neuroleptic-sensitive behavioral disorders.

Neurons contain abundant subsets of highly stable microtubules that resist depolymerizing conditions such as exposure to the cold. Stable microtubules are thought to be essential for neuronal development, maintenance, and function. Previous work has indicated an important role of the microtubule-associated protein STOP in the induction of microtubule cold stability. Here, we developed STOP null mice. These mice were devoid of cold-stable microtubules. In contrast to our expectations, STOP-/- mice had no detectable defects in brain anatomy but showed synaptic defects, with depleted synaptic vesicle pools and impaired synaptic plasticity, associated with severe behavioral disorders. A survey of the effects of psychotropic drugs on STOP-/- mice behavior showed a remarkable and specific effect of long-term administration of neuroleptics in alleviating these disorders. This study demonstrates that STOP is a major factor responsible for the intriguing stability properties of neuronal microtubules and is important for synaptic plasticity. Additionally, STOP-/- mice may yield a pertinent model for study of neuroleptics in illnesses such as schizophrenia, currently thought to result from synaptic defects.

Microtubule cold stability in supporting cells of the gerbil auditory sensory epithelium: correlation with tubulin post-translational modifications.

Sensory cells in the organ of Corti exhibit loose microtubule networks enriched in tyrosinated tubulin, whereas supporting cells have bundled microtubules containing post-translationally modified tubulin. The tubulin isoform distribution suggests that the microtubules in sensory cells are dynamic and those in supporting cells are stable. To test this, microtubule resistance to cold-induced depolymerization was examined by using immunocytochemical methods and antibodies to post-translationally modified tubulins. Microtubule labelling in cochleas perfused/immersed at room temperature was identical to that in previous studies of untreated cochleas. However, the microtubule patterns of perfused/immersed specimens were changed in cold-treated cochleas. Microtubules were no longer detected with antibodies to alpha- and tyrosinated tubulin in sensory cells from specimens exposed to cold, indicating their disassembly. Supporting cells in the same specimens showed almost total loss of detyrosinated and polyglutamylated tubulin in the middle and apical cochlear turns, and reduced labelling in the basal-most turn. Probing for alpha-, nontyrosinatable, acetylated and glycylated tubulin yielded decreased and sometimes patchy staining but these isoforms were observed even when detyrosinated and polyglutamylated tubulins were absent. The results indicate that sensory cells in the gerbil auditory sensory epithelium contain only cold-sensitive microtubules. In contrast, supporting cells possess a substantial subset of cold-stable microtubules, providing structural support to the vibratory sensory organ required for hearing.

Cold acclimation can induce microtubular cold stability in a manner distinct from abscisic acid.

The response of cortical microtubules to low temperature was investigated for the Chinese winter wheat (Triticum aestivum L.) cultivar Jing Nong 934. Microtubules in the cortex of the root elongation zone disassembled rapidly in response to a cold shock of -7 degrees C and reassembled upon rewarming to 25 degrees C. The microtubules acquired resistance against this cold shock in response to cold acclimation in chilling, but non-freezing, temperature or after a treatment with abscisic acid (ABA). Cold acclimation and ABA differed with respect to the appearance of microtubules: fine, transverse strands were observed after cold acclimation, whereas ABA produced steeply oblique microtubule bundles. The findings are discussed in terms of an ABA-independent pathway for acquired cold stability of microtubules.

Increase in cold-stability of microtubules by cold adaptation of Carassius cells in situ and in vitro

Increase in cold-stability of microtubules by cold adaptation of Carassius cells in situ and in vitro

Nonneuronal isoforms of STOP protein are responsible for microtubule cold stability in mammalian fibroblasts.

A number of cycling mammalian cells, such as NIH 3T3, contain abundant subsets of cold-stable microtubules. The origin of such microtubule stabilization in nonneuronal cells is unknown. We have previously described a neuronal protein, stable tubule-only polypeptide (STOP), that binds to microtubules and induces cold stability. We find that NIH 3T3 fibroblasts contain a major 42-kDa isoform of STOP (fibroblastic STOP, F-STOP). F-STOP contains the central repeats characteristic of brain STOP but shows extensive deletions of N- and C-terminal protein domains that are present in brain STOP. These deletions arise from differences in STOP RNA splicing. Despite such deletions, F-STOP has full microtubule stabilizing activity. F-STOP accumulates on cold-stable microtubules of interphase arrays and is present on stable microtubules within the mitotic spindle of NIH 3T3 cells. STOP inhibition by microinjection of affinity-purified STOP central repeat antibodies into NIH 3T3 cells abolishes both interphase and spindle microtubule cold stability. Similar results were obtained with Rat2 cells. These results show that STOP proteins have nonneuronal isoforms that are responsible for the microtubule cold stability observed in mammalian fibroblasts.

Cloning, expression, and properties of the microtubule-stabilizing protein STOP.

Nerve cells contain abundant subpopulations of cold-stable microtubules. We have previously isolated a calmodulin-regulated brain protein, STOP (stable tubule-only polypeptide), which reconstitutes microtubule cold stability when added to cold-labile microtubules in vitro. We have now cloned cDNA encoding STOP. We find that STOP is a 100.5-kDa protein with no homology to known proteins. The primary structure of STOP includes two distinct domains of repeated motifs. The central region of STOP contains 5 tandem repeats of 46 amino acids, 4 with 98% homology to the consensus sequence. The STOP C terminus contains 28 imperfect repeats of an 11-amino acid motif. STOP also contains a putative SH3-binding motif close to its N terminus. In vitro translated STOP binds to both microtubules and Ca2+-calmodulin. When STOP cDNA is expressed in cells that lack cold-stable microtubules, STOP associates with microtubules at 37 degrees C, and stabilizes microtubule networks, inducing cold stability, nocodazole resistance, and tubulin detyrosination on microtubules in transfected cells. We conclude that STOP must play an important role in the generation of microtubule cold stability and in the control of microtubule dynamics in brain.

Microtubules stability in human peripheral nerves

We examined the cold stability of microtubules (MT) in biopsied sural nerves. In control nerves, cold treatment reduced the density of MT in unmyelinated axons. The proportion of cold-insoluble MT was approximately 30% by morphological study. By contrast, the extraction study using essentially the same nerves showed that the ratio of cold-insoluble tubulin was 60–75%. These findings suggest the existence of a subpopulation of cold-stable tubulin which may not make up MT. However, in 2 patients with acute dysautonomia the proportion of cold-stable MT or tubulin was decreased compared to that in controls in both the morphological and extraction study. This suggests the altered local process involving MT in primary axonal degeneration.

Rhazinilam mimics the cellular effects of taxol by different mechanisms of action

We have investigated the effects of the microtubule poison rhazinilam on microtubule assembly in vivo and in vitro. In mammalian cells, rhazinilam mimics the effects of taxol and leads to microtubule bundles, multiple asters, and microtubule cold stability. In vitro, rhazinilam protected preassembled microtubules from cold-induced disassembly, but not from calcium ion-induced disassembly. Moreover, both at 0 degrees C and at 37 degrees C, rhazinilam induced the formation of anomalous tubulin assemblies (spirals). This process was prevented by maytansine and vinblastine, but not by colchicine. Preferential saturable and stoichiometric binding of radioactive rhazinilam to tubulin in spirals was observed with a dissociation constant of 5 microM. This binding was abolished in the presence of vinblastine and maytansine. In contrast, specific binding of radioactive rhazinilam to tubulin assembled in microtubules was undetectable. These results demonstrate that rhazinilam alters microtubule stability differently than taxol, and that the overall similar effects of rhazinilam and taxol on the cellular cytoskeleton are the consequence of two distinct mechanisms of action at the molecular level.

Posttranslational modifications and assembly characteristics of goldfish tubulin.

Cell-free extracts from goldfish brain, ovarian follicles, testes and the cell line, ATCC CCL-71, were analyzed for posttranslationally modified tubulins. All samples, with the exception of that from brain where the reverse was true, contained more tyrosinated than detyrosinated alpha-tubulin. Additionally, extracts from brain and testes exhibited acetylated alpha-tubulin whereas this isoform was not visualized on blots of cell-free preparations from follicles and CCL-71 cells. Assembly of brain and ovary tubulin was induced with taxol. Brain tubulin, partially purified through three cycles of assembly/disassembly was associated with a variety of putative microtubule-associated proteins (MAPs), most of which had a high molecular mass. There were very few cold stable microtubules in brain preparations whereas, for ovary, purification of tubulin was hampered by significant losses of cold stable polymer. Comparison of brain and ovary showed there was no correlation between the extent of alpha-tubulin detyrosination or acetylation and cold stability of microtubules. Moreover, cycled tubulin from ovary contained acetylated tubulin even though this was not observed on blots of cell-free extracts from ovary or from follicles. Cultured goldfish cells contained extensive arrays of microtubules, many of which originated from discrete organizing centers. The results reveal the widespread distribution of posttranslationally modified tubulins in goldfish tissues, the differing assembly/disassembly characteristics of tubulin from brain versus ovary, and the presence of putative neural MAPs, mostly with a high molecular mass.

Induction of Cold Stability of Microtubules in Cultured Tobacco Cells

In suspension-cultured tobacco (Nicotiana tabacum) cells, we have often encountered cold-stable microtubules (MTs). The cold-stable MTs were found in the pelleted fraction of tobacco cell homogenates. These cold-stable MTs were shown to be accompanied by unidentified filamentous structures that extended along part of their length. However, during the early hours in culture such cold-stable MTs were never observed. They were detectable from 120 h after the beginning of subculture and then their numbers increased gradually. The number of cells with cold-stable MTs eventually accounted for more than 95% of the total population of cells at the stationary phase of culture. The rapid loss of cold stability of MTs occurred when such cells were transferred to fresh medium for subculture. However, if the fresh medium was supplemented with once-used medium, the cold stability of MTs was retained. The active agent in the medium appeared to be of low molecular weight and to be heat resistant. A similar activity was detected in a pectin hydrolyzate. When an inhibitor of protein kinase, either 6-dimethylaminopurine or staurosporin, was added to the cells at an early stage of culture, when cold-stable MTs were normally completely absent, most cells acquired cold-stable MTs. It appears that acquisition or loss of cold stability of MTs in tobacco cells is regulated by the action of a kinase/phosphatase or a phosphorylation/dephosphorylation system on some MT protein(s), such as a cold stabilizer of MTs, some unidentified MT-associated filamentous structure, or even tubulin itself.

Cis-Diamminedichloroplatinum (II) (cisplatin) alters microtubule assembly dynamics

Cisplatin is an highly effective chemotherapeutic agent which produces a cumulative dose-limiting peripheral neuropathy. In this study, a possible role for microtubule abnormalities in cisplatin-induced toxicity was explored. CD rats (300 g) were injected daily ip with five doses of cisplatin (2 mg/kg) or every other day ip with two doses of cisplatin (10 mg/kg). The day after the last dose, the rats were killed and tubulin was purified from their testes. The maximal rate of cold-induced microtubule disassembly was consistently slower for testis tubulin purified from cisplatin-treated rats compared with control rats. Overnight in vitro coincubation of polymerized bovine brain tubulin with cisplatin followed by a purifying cycle of assembly and disassembly yielded tubulin capable of forming morphologically normal but short microtubules (average length: cisplatin-treated, 2.5 μm; control, 3.7 μm). Cisplatin coincubation markedly reduced the rate of cold-induced microtubule disassembly, producing a half-maximal effect at approximately 0.3 m m. The cold stability of cisplatin-treated microtubules could be partially reversed by diethyldithiocarbamate. Carboplatin, a cisplatin analog which does not cause clinical peripheral neuropathy, was less capable of producing microtubule disassembly abnormalities. These findings demonstrate the ability of cisplatin to alter microtubule disassembly by direct tubulin modification, an abnormality which may contribute to cisplatin-induced peripheral neuropathy.