Human TUBB3 Mutations Perturb Microtubule Dynamics, Kinesin Interactions, and Axon Guidance (original) (raw)
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The emerging role of the tubulin code: From the tubulin molecule to neuronal function and disease
Cytoskeleton, 2016
Across different cell types and tissues, microtubules are assembled from highly conserved dimers of a-and b-tubulin. Despite their highly similar structures, microtubules have functional heterogeneity, generated either by the expression of different tubulin genes, encoding distinct isotypes, or by posttranslational modifications of tubulin. This genetically encoded and posttranslational generated heterogeneity of tubulinthe "tubulin code"-has the potential to modulate microtubule structure, dynamics, and interactions with associated proteins. The tubulin code is therefore believed to regulate microtubule functions on a cellular and sub-cellular level. This review highlights the importance of the tubulin code for tubulin structure, as well as on microtubule dynamics and functions in neurons. It further summarizes recent developments in the understanding of mutations in tubulin genes, and how they are linked to neurodegenerative and neurodevelopmental disorders. The current advances in the knowledge of the tubulin code on the molecular and the functional level will certainly lead to a better understanding of how complex signaling events control microtubule functions, especially in cells of the nervous system. V
Mutations in the β-Tubulin Gene TUBB5 Cause Microcephaly with Structural Brain Abnormalities
Cell Reports, 2012
The formation of the mammalian cortex requires the generation, migration, and differentiation of neurons. The vital role that the microtubule cytoskeleton plays in these cellular processes is reflected by the discovery that mutations in various tubulin isotypes cause different neurodevelopmental diseases, including lissencephaly (TUBA1A), polymicrogyria (TUBA1A, TUBB2B, TUBB3), and an ocular motility disorder (TUBB3). Here, we show that Tubb5 is expressed in neurogenic progenitors in the mouse and that its depletion in vivo perturbs the cell cycle of progenitors and alters the position of migrating neurons. We report the occurrence of three microcephalic patients with structural brain abnormalities harboring de novo mutations in TUBB5 (M299V, V353I, and E401K). These mutant proteins, which affect the chaperonedependent assembly of tubulin heterodimers in different ways, disrupt neurogenic division and/or migration in vivo. Our results provide insight into the functional repertoire of the tubulin gene family, specifically implicating TUBB5 in embryonic neurogenesis and microcephaly.
Developmental Neuroscience, 2008
duced proportion of tyrosinated microtubules, we analyzed the possible interaction between MAP1B and tubulin tyrosine ligase. Our results show that these proteins indeed interact and that the interaction is not affected by MAP1B phosphorylation. Additionally, neurons lacking MAP1B, when exposed to drugs that reversibly depolymerize microtubules, do not fully recover tyrosinated microtubules upon drug removal. These results suggest that MAP1B regulates tyrosination of ␣ -tubulin in neuronal microtubules. This regulation may be important for general processes involved in nervous system development such as axonal guidance and neuronal migration.
Isolation of Functional Tubulin Dimers and of Tubulin-Associated Proteins from Mammalian Cells
Current biology : CB, 2016
The microtubule (MT) cytoskeleton forms a dynamic filamentous network that is essential for many processes, including mitosis, cell polarity and shape, neurite outgrowth and migration, and ciliogenesis [1, 2]. MTs are built up of α/β-tubulin heterodimers, and their dynamic behavior is in part regulated by tubulin-associated proteins (TAPs). Here we describe a novel system to study mammalian tubulins and TAPs. We co-expressed equimolar amounts of triple-tagged α-tubulin and β-tubulin using a 2A "self-cleaving" peptide and isolated functional fluorescent tubulin dimers from transfected HEK293T cells with a rapid two-step approach. We also produced two mutant tubulins that cause brain malformations in tubulinopathy patients [3]. We then applied a paired mass-spectrometry-based method to identify tubulin-binding proteins in HEK293T cells and describe both novel and known TAPs. We find that CKAP5 and the CLASPs, which are MT plus-end-tracking proteins with TOG(L)-domains [4], b...
γ-Tubulin 2 Nucleates Microtubules and Is Downregulated in Mouse Early Embryogenesis
PLoS ONE, 2012
c-Tubulin is the key protein for microtubule nucleation. Duplication of the c-tubulin gene occurred several times during evolution, and in mammals c-tubulin genes encode proteins which share ,97% sequence identity. Previous analysis of Tubg1 and Tubg2 knockout mice has suggested that c-tubulins are not functionally equivalent. Tubg1 knockout mice died at the blastocyst stage, whereas Tubg2 knockout mice developed normally and were fertile. It was proposed that c-tubulin 1 represents ubiquitous c-tubulin, while c-tubulin 2 may have some specific functions and cannot substitute for c-tubulin 1 deficiency in blastocysts. The molecular basis of the suggested functional difference between c-tubulins remains unknown. Here we show that exogenous c-tubulin 2 is targeted to centrosomes and interacts with c-tubulin complex proteins 2 and 4. Depletion of c-tubulin 1 by RNAi in U2OS cells causes impaired microtubule nucleation and metaphase arrest. Wild-type phenotype in c-tubulin 1-depleted cells is restored by expression of exogenous mouse or human c-tubulin 2. Further, we show at both mRNA and protein levels using RT-qPCR and 2D-PAGE, respectively, that in contrast to Tubg1, the Tubg2 expression is dramatically reduced in mouse blastocysts. This indicates that c-tubulin 2 cannot rescue c-tubulin 1 deficiency in knockout blastocysts, owing to its very low amount. The combined data suggest that c-tubulin 2 is able to nucleate microtubules and substitute for c-tubulin 1. We propose that mammalian c-tubulins are functionally redundant with respect to the nucleation activity.
Cellular regulation of microtubule organization
The Journal of cell biology, 1984
Microtubules are constituents of axonemes, mitotic spindles, and elaborate arrays in interphase cells, and, with intermediate filaments and microfilaments, are among the most prevalent structures visualized in the cytomatrix (22, 44). With the exception ofthe A microtubule ofcilia and flagella, the lattice geometry ofmicrotubules is highly conserved. However, each of the major subunits of microtubules, a-and a-tubulin, shows heterogeneity. The number ofa-and f3-tubulin subspecies differs among tissues and organisms, and a number of types of analysis are used to examine how these tubulin variants are related to specific cell functions (1, 9-11, 33, 40). Investigations of the number and complexity of genes coding for these polypeptides have also been initiated (see reference 13 for review). However, the mechanisms that regulate the posttranslational compartmentalization of subunits, the spatial and temporal assembly of subunits into microtubules, and the integration of microtubules in various cellular events are still largely unknown. There are many levels at which the formation and organization of microtubules might be determined. A postulate originating from early analyses of mitotic spindle formation (32) was that a pool of subunits existed in equilibrium with formed microtubules; increases in the subunit concentration could therefore result in a net increase in polymer. With few exceptions, however, a rapid increase in the total tubulin pool does not appear to occur before the elaboration of more extensive microtubule arrays. For example, our studies (42, 50) have demonstrated that mouse neuroblastoma cells possessing microtubule-filled neurites contain four to five times more tubulin polymer than rounded, nondifferentiated cells, but the total tubulin content of these two cell types is the same. On the basis of volume calculations, the equilibrium concentration of subunits in the nondifferentiated cells is at least twice that in differentiated cells. Data such as this indicate that a simple equilibrium between subunit and polymer cannot account for the changes in microtubule formation coordinated with certain cellular events. In addition, recent findings show that an increase in the subunit concentration in cells, brought about either by drug treatment (15) or injection of tubulin (16), results in a depression of tubulin synthesis and the loss of tubulin mRNA. These data suggest that cells autoregulate the total tubulin pool and that this may be effected by "monitoring" of the monomer concentration (14).
Post-translational modifications of tubulin: pathways to functional diversity of microtubules
Trends in Cell Biology, 2015
Tubulin and microtubules are subject to a remarkable number of posttranslational modifications. Understanding the roles these modifications play in determining functions and properties of microtubules has presented a major challenge that is only now being met. Many of these modifications are found concurrently, leading to considerable diversity in cellular microtubules, which varies with development, differentiation, cell compartment and cell cycle. We now know that posttranslational modifications of tubulin affect not only the dynamics of the microtubules, but also their organization and interaction with other cellular components. Many early suggestions of how posttranslational modifications affect microtubules have been replaced with new ideas and even new modifications as our understanding of cellular microtubule diversity comes into focus.
Annals of neurology, 2017
Our goal was to define the genetic cause of the profound hypomyelination in the taiep rat model and determine its relevance to human white matter disease. Based on previous localization of the taiep mutation to rat chromosome 9, we tested if the mutation resided within the Tubb4a (β-tubulin 4A) gene, since mutations in the TUBB4A gene have been described in patients with CNS hypomyelination. To determine whether accumulation of microtubules led to progressive demyelination we analyzed the spinal cord and optic nerves of 2 year old rats by light and electron microscopy. Cerebral white matter from a patient with TUBB4A Asn414Lys mutation and MRI evidence of severe hypomyelination was studied similarly. As the taiep rat ages there is progressive loss of myelin in the brain and dorsal column of the spinal cord associated with increased oligodendrocyte numbers with accumulation of microtubules. This accumulation involved the entire cell body and distal processes of oligodendrocytes but t...
A mutation in theTubb4agene leads to microtubule accumulation with hypomyelination and demyelination
Annals of Neurology, 2017
Objective-Our goal was to define the genetic cause of the profound hypomyelination in the taiep rat model and determine its relevance to human white matter disease. Methods-Based on previous localization of the taiep mutation to rat chromosome 9, we tested if the mutation resided within the Tubb4a (β-tubulin 4A) gene, since mutations in the TUBB4A gene have been described in patients with CNS hypomyelination. To determine whether accumulation of microtubules led to progressive demyelination we analyzed the spinal cord and optic nerves of 2 year old rats by light and electron microscopy. Cerebral white matter from a patient with TUBB4A Asn414Lys mutation and MRI evidence of severe hypomyelination was studied similarly. Results-As the taiep rat ages there is progressive loss of myelin in the brain and dorsal column of the spinal cord associated with increased oligodendrocyte numbers with accumulation of microtubules. This accumulation involved the entire cell body and distal processes of oligodendrocytes but there was no accumulation of microtubules in axons. A single point mutation