Disintegration Of Microtubules Research Articles

Lifetime of biomolecules in polymer-based hybrid nanodevices

Prolonging the lifetime of biomolecules in their functional states is critical for manyapplications where biomolecules are integrated into synthetic materials or devices.A simplified molecular shuttle system, which consists of fluorescently labelledmicrotubules propelled by kinesin motor proteins bound to the surface of a flowcell,served here as a model system to probe the lifetime of a hybrid device. In thissystem, the functional decay can easily be assayed by utilizing optical microscopy todetect motility and disintegration of microtubules. We found that the lifetimes ofthese hybrid systems were mainly limited by the stability of microtubules (MTs),rather than of kinesin. To determine the biocompatibility of polymers widelyused in microfabrication, we assembled flowcells with glass bottom surfaces andcovers fabricated from glass, poly(urethane) (PU), poly(methyl-methacrylate)(PMMA), poly(dimethylsiloxane) (PDMS) and ethylene-vinyl alcohol copolymer(EVOH). Without illumination, only PU had a substantial negative impact on MTstability, while PMMA, PDMS and EVOH showed stabilities comparable to glass.Under the influence of light, however, the MTs degraded rapidly in the presence ofPDMS or PMMA, even in the presence of oxygen scavengers. A similar effect wasobserved on glass if oxygen scavengers were not added to the medium. Strongbleaching of the fluorophores was again only found on the polymer substratesand photobleaching coincided with an accelerated depolymerization of the MTs.

Ultrastructure of Purkinje cell perikarya and their dendritic processes in the rat cerebellar cortex in experimental encephalopathy induced by chronic application of valproate.

Long-term intragastric administration of the antiepileptic drug sodium valproate (Vuprol Polfa) to rats for 1, 3, 6, 9 and 12 months, once daily at the effective dose of 200 mg/kg body weight showed morphological evidence of encephalopathy, manifested by numerous nonspecific changes within Purkinje cell perikarya and their dendritic processes. The first ultrastructural abnormalities appeared after 3 months. They became more severe in animals with longer survival and were most pronounced after 12 months. The changes were maintained both 1 and 3 months after drug withdrawal. Mitochondria of Purkinje cell perikarya were most severely affected. Damage to mitochondria was accompanied by disintegration and fragmentation of granular endoplasmic reticulum, dilation of channels and cisterns of Golgi apparatus, enlargement of smooth endoplasmic reticulum elements including submembranous cisterns, and accumulation of profuse lipofuscin deposits. Frequently, Purkinje cells appeared as dark ischemic neurones, with focally damaged cellular membrane and features of disintegration. Swollen Bergmann's astrocytes were seen among damaged Purkinje cells or at the site of their loss. The general pattern of submicroscopic alterations of Purkinje cell perikarya suggested severe disorders in several intercellular biochemical extents, including inhibition of oxidative phosphorylation and abnormal protein synthesis, both of which could lead to lethal damage. Ultrastructural abnormalities within dendrites were characterized by damage to elements of smooth endoplasmic reticulum, which was considerably enlarged, with formation of large vacuolar structures situated deep in the dendroplasm. Mitochondrial lesions and alterations in cytoskeletal elements--disintegration of microtubules or even their complete loss--were also observed. The general pattern of abnormalities within the organelles and cytoskeletal elements of dendritic processes in Purkinje cells in the VPA chronic experimental model imply that there are disturbances in detoxication processes. Furthermore these changes were irreversible, as they were maintained after drug withdrawal.

Interaction of Tau with the Neural Membrane Cortex Is Regulated by Phosphorylation at Sites That Are Modified in Paired Helical Filaments

The axonal microtubule-associated phosphoprotein tau interacts with neural plasma membrane (PM) components during neuronal development (Brandt, R., Léger, J., and Lee, G. (1995) J. Cell Biol. 131, 1327-1340). To analyze the mechanism and potential regulation of tau's PM association, a method was developed to isolate PM-associated tau using microsphere separation of surface-biotinylated cells. We show that tau's PM association requires an intact membrane cortex and that PM-associated tau and cytosolic tau are differentially phosphorylated at sites detected by several Alzheimer's disease (AD) diagnostic antibodies (Ser(199)/Ser(202), Thr(231), and Ser(396)/Ser(404)). In polar neurons, the association of endogenous tau phosphoisoforms with the membrane cortex correlates with an enrichment in the axonal compartment. To test for a direct effect of AD-specific tau modifications in determining tau's interactions, a phosphomutant that simulates an AD-like hyperphosphorylation of tau was produced by site-directed mutagenesis of Ser/Thr residues to negatively charged amino acids (Glu). These mutations completely abolish tau's association with the membrane cortex; however, the construct retains its capability to bind to microtubules. The data suggest that a loss of tau's association with the membrane cortex as a result of phosphorylation at sites that are modified during disease contributes to somatodendritic tau accumulation, axonal microtubule disintegration, and neuronal death characteristic for AD.

Colocalization of microtubules and mitochondria in the yeast Schizosaccharomyces japonicus var. versatilis.

Both living and fixed cells of Schizosaccharomyces japonicus var. versatilis showed thread-like mitochondria when studied by phase-contrast and fluorescence microscopy. In the interphase cells, mitochondria extended from pole to pole and converged towards the growing tips. The mitochondrial threads did not disrupt but persisted during mitosis and, subsequently, their bundle was split between the two daughter cells by a concentrically growing septum. Mitochondria in the interphase cells were accompanied by cytoplasmic microtubules. These disappeared during mitosis and, instead, spindle microtubules were formed in the nucleus. The cytoplasmic microtubules reappeared after anaphase B, again in coaligment with mitochondria. Protoplasting as well as the action of microtubule inhibitors methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate (benomyl) and 2-methylbenzimidazole (MBC) resulted in rapid disintegration of microtubules and, suprisingly, also in disruption of mitochondria into small bodies. Removal of the inhibitors or a short regeneration of protoplasts allowed both the cytoplasmic microtubules and the thread-like mitochondria to reaggregate into the original pattern. Cytochalasin D treatment caused a complete disintegration of actin filaments, while the cytoplasmic microtubules and mitochondria remained intact. These findings of a transient close association of mitochondria and microtubules and their relative independence of the arrangement of actin filaments suggest that microtubules, but not actin cables, form supports for positioning or movement of mitochondria along the cylindrical cells. The persistence of mitochondria in the cell centre during mitosis may be accounted for by the fact that disrupted microtubules fail to provide support for mitochondrial movement towards the cell poles.

Golgi electron microscopic study of the cerebral cortex after transient cerebral ischemia and reperfusion in the gerbil

Fine structures of defined neurons and their dendritic processes were studied in the cerebral cortex of gerbil brains by using Golgi electron microscopy during progressive cerebral ischemia for 10 and 20 min and after reperfusion for up to 72 h following transient ischemia for 20 min. The periphery of ascending dendrites of the vulnerable neurons in layers III and Vb became distended immediately after ischemia with swollen mitochondria and disintegrated microtubules, but the proximal portion of the same dendrites remained unchanged. After reperfusion for 6 h, distension of the dendroplasm of the impregnated dendrites in layer I receded, but the proximal portion of the same dendrites showed indentation caused by swollen astrocytic processes and derangement of microtubules inside. Polyribosomes in most neuronal perikarya were disaggregated, but severe neuronal damage was rarely found among those neuronal cell bodies impregnated by the Golgi method. Recovery with reaggregation of polyribosomes and realignment of microtubules was more clearly observed after reperfusion for 24 h and thereafter in impregnated neurons. These results indicated that impregnation during progressive ischemia occurred in many neurons with progressive structural damage but that impregnation during reperfusion occurred in a limited number of neurons with limited damage, allowing us to observe the recovery process, and that neuronal derangement in the dendrosomatic direction initially occurred both in the irreversibly damaged neurons and in the reversibly damaged ones. It is possible that disintegration of microtubules and the resulting disruption of dendritic transport may contribute to subsequent development of delayed neuronal death, if the recovery process does not take place promptly. Golgi electron microscopy is useful for ultrastructural investigation of defined neurons and their dendrites together and may be applicable for investigation of selected neuropathologic conditions.

Immunoelectron microscopic investigation of creatine kinase BB-isoenzyme after cerebral ischemia in gerbils.

Alteration of creatine kinase BB-isoenzyme (CK-BB) was investigated in the vulnerable CA1 region of the hippocampus of ischemic and postischemic gerbil brains using immunoelectron microscopy. CK-BB existed in the neuronal perikarya, dendrites and axons as well as in astroglias in the normal gerbil brain. Immunocytochemical reaction products were associated with microtubules and polyribosomes. Propagation of ischemic and postischemic damage with disintegration of microtubules was observed in the dendro-somatic direction in neurons, which progressed in parallel with dispersion and loss of the immunocytochemical reaction for CK-BB in the dendroplasm. After reperfusion for longer than 24 h, CK-BB was also observed in the extracellular space. The present result supported the notion that loss of the immunohistochemical reaction for CK-BB which has been observed by light microscopy after cerebral ischemia, was at least partly due to dispersion of this enzyme caused by disintegration of microtubules and extracellular leakage of this enzyme, although other processes, including degradation of CK-BB per se, were also possible. The loss of CK-BB from the neuronal structure may delay the recovery from ischemic damage and may eventually lead to neuronal death.

The Sliding of the Fibrous Sheath through the Axoneme Proximally Together with Microtubule Extrusion

When activated mouse sperm were treated with Triton X-100 and dithiothreitol at pH 9.0, the plasma membrane and mitochondrial sheath of the sperm flagella were removed. These mitochondrion-free demembranated sperm flagella were perfused with Mg-ATP and trypsin to induce the sliding disintegration of microtubules from the axoneme. We observed that when the doublet microtubules associated with outer dense fibers extruded from the axoneme, the fibrous sheath (FS) was pulled proximally to the connecting piece from the annulus of the sperm flagellum accompanying the extrusion of microtubules. Furthermore, the FS sliding was cAMP-dependent and the maximal sliding velocity was about 3.7 ± 0.1 μm/s in the presence of 1.0 mM ATP at 37 + 1°C. Since the FS projected inward and attached opposite doublet microtubules 3 and 8 in the principal piece, our observation showed the possibility that doublet 3 and/or 8 participated in FS sliding.

Electron microscopic investigation of the cerebral cortex after cerebral ischemia and reperfusion in the gerbil

Prompt dendritic damage has been observed in the hippocampus of the gerbil brain after transient cerebral ischemia. In the present study, we studied the frontoparietal cortex of the gerbil brain electron microscopically after brief bilateral carotid occlusion to assess the vulnerability of dendritic processes. After ischemia for 5 min, there was swelling of the periphery of dendrites accompanied by swelling of mitochondria, cytoplasmic vacuolation and disintegration of microtubules in layer I, which spread to layer III after ischemia for 20 min. After reperfusion for 3–24 h following ischemia for 20 min, swelling in the periphery of dendrites and of mitochondria inside receded but vacuole formation and disintegration of microtubules propagated proximally. In neuronal perikarya, polyribosomal disaggregation was observed after ischemia for 20 min and persisted thereafter, while fragmentation of rough endoplasmic reticulum (ER) and microvacuolation occured after reperfusion for 3 h. Electron-dense clumping of neuronal perikarya was observed after reperfusion for 6 h particularly in layers III and Vb, which increased in number for up to 72 h. The observed progressive damage in dendrites may be common to neurons vulnerable to cerebral ischemia and may significantly contribute to development of delayed neuronal death.

Immunoelectron microscopic study of tubulin and microtubule-associated proteins after transient cerebral ischemia in gerbils.

Differential vulnerability of microtubule components to cerebral ischemia has been reported previously. We investigated the disintegration of microtubules using immunoelectron microscopy for alpha-tubulin and microtubule-associated protein 1A and 2 (MAP1A and 2). Mongolian gerbils were subjected to bilateral carotid occlusion for 10 to 30 min and reperfusion for up to 72 h following ischemia for 10 min. After ischemia for 10 min, some dendrites in the stratum moleculare of the subiculum-CA1 region lost immunoreaction products for alpha-tubulin and MAPs. Loss of the reaction products spread to the medial CA1 region during progressive ischemia for 30 min. In some dendrites, electron-dense precipitates for MAPs were dispersed in the dendritic cytoplasm with little reaction product on microtubules and without alteration of the reaction for alpha-tubulin. After recirculation, loss of electron-dense precipitates for alpha-tubulin and MAPs, as well as disintegration of microtubules, propagated further to the medial CA1 region and to the proximal dendrites. The present study demonstrated prompt disintegration of microtubules with rapid disappearance of the reaction for MAPs which seemed to be caused by detachment of MAPs from the microtubule cores.

Cerebral ischemia in the gerbil: Transmission electron microscopic and immunoelectron microscopic investigation

Progression of cerebral ischemia from 5 min to 3 h after occlusion of a common carotid artery was investigated in the subiculum-CA1 region of the hippocampus of the gerbil by transmission electron microscopic and immunoelectron microscopic technique. The earliest change was found after 5 min in the periphery of the apical dendrites in the striatum moleculare, where mitochondrial swelling and disintegration of microtubules were clearly seen inside swollen dendritic processes. After ischemia for 10 min, similar abnormalities were observed in the more proximal part of the apical dendrites, and the basal dendrites also became similarly affected. After ischemia for 30 min to 1 h, the pyramidal cell bodies showed mitochondrial swelling, distension of endoplasmic reticulum and disaggregation of polyribosomes. The immunoelectron microscopic procedure for tubulin revealed irregularity of reaction products associated with microtubules after ischemia for 5 min in the dendritic terminals in the striatum moleculare and in the radiatum after ischemia for 10 min. Reaction products in the pyrimidal cell bodies became sparse after ischemia for 30 min to 1 h. The present investigation revealed early onset of ischemic damage in the dendritic terminals and subsequent proximal extension, with disintegration of microtubules and mitochondrial swelling.

The formation of annulated lamellae induced by the disintegration of microtubules.

Investigations with different 'antitubulins' have shown that different cell types in culture response to prolonged treatment with the formation of annulated lamellae. These annulated lamellae are possibly derived from previously accumulated smooth endoplasmic profiles. A hypothetical explanation would be that annulated lamellae (and the nuclear membrane) are sites of tubulin synthesis or microtubular polymerization. Their appearance after induced microtubule disintegration could then be viewed upon as a reactive hyperproduction of such sites.