Academic literature on the topic 'Microtubule stabilization'

Increased microtubule density, for which microtubule stabilization is one potential mechanism, causes contractile dysfunction in cardiac hypertrophy. After microtubule assembly, α-tubulin undergoes two, likely sequential, time-dependent posttranslational changes: reversible carboxy-terminal detyrosination (Tyr-tubulin ↔ Glu-tubulin) and then irreversible deglutamination (Glu-tubulin → Δ2-tubulin), such that Glu- and Δ2-tubulin are markers for long-lived, stable microtubules. Therefore, we generated antibodies for Tyr-, Glu-, and Δ2-tubulin and used them for staining of right and left ventricular cardiocytes from control cats and cats with right ventricular hypertrophy. Tyr- tubulin microtubule staining was equal in right and left ventricular cardiocytes of control cats, but Glu-tubulin and Δ2-tubulin staining were insignificant, i.e., the microtubules were labile. However, Glu- and Δ2-tubulin were conspicuous in microtubules of right ventricular cardiocytes from pressure overloaded cats, i.e., the microtubules were stable. This finding was confirmed in terms of increased microtubule drug and cold stability in the hypertrophied cells. In further studies, we found an increase in a microtubule binding protein, microtubule-associated protein 4, on both mRNA and protein levels in pressure-hypertrophied myocardium. Thus, microtubule stabilization, likely facilitated by binding of a microtubule-associated protein, may be a mechanism for the increased microtubule density characteristic of pressure overload cardiac hypertrophy.

Axon formation is the initial step in establishing neuronal polarity. We examine here the role of microtubule dynamics in neuronal polarization using hippocampal neurons in culture. We see increased microtubule stability along the shaft in a single neurite before axon formation and in the axon of morphologically polarized cells. Loss of polarity or formation of multiple axons after manipulation of neuronal polarity regulators, synapses of amphids defective (SAD) kinases, and glycogen synthase kinase-3β correlates with characteristic changes in microtubule turnover. Consistently, changing the microtubule dynamics is sufficient to alter neuronal polarization. Application of low doses of the microtubule-destabilizing drug nocodazole selectively reduces the formation of future dendrites. Conversely, low doses of the microtubule-stabilizing drug taxol shift polymerizing microtubules from neurite shafts to process tips and lead to the formation of multiple axons. Finally, local stabilization of microtubules using a photoactivatable analogue of taxol induces axon formation from the activated area. Thus, local microtubule stabilization in one neurite is a physiological signal specifying neuronal polarization.

We have developed a method to distinguish microtubule associated protein (MAP)-containing regions from MAP-free regions within a microtubule, or within microtubule sub-populations. In this method, we measure the MAP-dependent stabilization of microtubule regions to dilution-induced disassembly of the polymer. The appropriate microtubule regions are identified by assembly in the presence of [3H]GTP, and assayed by filter trapping and quantitation of microtubule regions that contain label. We find that MAPs bind very rapidly to polymer binding sites and that they do not exchange from these sites measurably once bound. Also, very low concentrations of MAPs yield measurable stabilization of local microtubule regions. Unlike the stable tubule only polypeptide (STOP) proteins, MAPs do not exhibit any sliding behavior under our assay conditions. These results predict the presence of different stability subclasses of microtubules when MAPs are present in less than saturating amounts. The data can readily account for the observed "dynamic instability" of microtubules through unequal MAP distributions. Further, we report that MAP dependent stabilization is quantitatively reversed by MAP phosphorylation, but that calmodulin, in large excess, has no specific influence on MAP protein activity when MAPs are on microtubules.

We previously transfected MAP2, tau and MAP1B cDNA into fibroblasts and have studied the effect of expression of these microtubule-associated proteins on microtubule organization. In this study, we examined some additional characteristics of microtubule bundles and arrays formed in fibroblasts transfected with these microtubule-associated proteins. It was found that microtubule bundles formed in MAP2c- or tau-transfected cells were stabilized against microtubule depolymerizing reagents and were enriched in acetylated alpha tubulin. When mouse MAP1B cDNA was expressed following transfection into COS cells, MAP1B was localized along microtubule arrays, but no extensive reorganization of microtubules such as bundle formation was observed, in agreement with our previous finding using HeLa and 3T3 cells. However, stabilization of microtubules was indicated: (a) microtubules in MAP1B-transfected cells were stabilized against a microtubule depolymerizing reagent, although stabilization was less efficient than that seen in MAP2c- or tau-transfected cells, and (b) microtubules in MAP1B-transfected cells were enriched in acetylated alpha tubulin. These results suggest that neuronal microtubule-associated proteins introduced into fibroblasts by cDNA transfection stabilize microtubules and affect the state of post-translational modification of tubulin.

The plant cortical microtubule array is a unique acentrosomal array that is essential for plant morphogenesis. To understand how this array is organized, we exploited the microtubule (+)-end tracking activity of two Arabidopsis EB1 proteins in combination with FRAP (fluorescence recovery after photobleaching) experiments of GFP-tubulin to examine the relationship between cortical microtubule array organization and polarity. Significantly, our observations show that the majority of cortical microtubules in ordered arrays, within a particular cell, face the same direction in both Arabidopsis plants and cultured tobacco cells. We determined that this polar microtubule coalignment is at least partially due to a selective stabilization of microtubules, and not due to a change in microtubule polymerization rates. Finally, we show that polar microtubule coalignment occurs in conjunction with parallel grouping of cortical microtubules and that cortical array polarity is progressively enhanced during array organization. These observations reveal a novel aspect of plant cortical microtubule array organization and suggest that selective stabilization of dynamic cortical microtubules plays a predominant role in the self-organization of cortical arrays.

Neuronal differentiation and function require extensive stabilization of the microtubule cytoskeleton. Neurons contain a large proportion of microtubules that resist the cold and depolymerizing drugs and exhibit slow subunit turnover. The origin of this stabilization is unclear. Here we have examined the role of STOP, a calmodulin-regulated protein previously isolated from cold-stable brain microtubules. We find that neuronal cells express increasing levels of STOP and of STOP variants during differentiation. These STOP proteins are associated with a large proportion of microtubules in neuronal cells, and are concentrated on cold-stable, drug-resistant, and long-lived polymers. STOP inhibition abolishes microtubule cold and drug stability in established neurites and impairs neurite formation. Thus, STOP proteins are responsible for microtubule stabilization in neurons, and are apparently required for normal neurite formation.

The acidic carboxy-terminal regions of alpha- and beta-tubulin subunits are currently thought to be centrally involved in microtubule stability and in microtubule association with a variety of proteins (MAPs) such as MAP2 and tau proteins. Here, pure tubulin microtubules were exposed to subtilisin to produce polymers composed of cleaved tubulin subunits lacking carboxy termini. Polymer exposure to subtilisin was achieved in buffer conditions compatible with further tests of microtubule stability. Microtubules composed of normal alpha-tubulin and cleaved beta-tubulin were indistinguishable from control microtubules with regard to resistance to dilution-induced disassembly, to cold temperature-induced disassembly and to Ca(2+)-induced disassembly. Microtubules composed of cleaved alpha- and beta-tubulins showed normal sensitivity to dilution-induced disassembly and to low temperature-induced disassembly, but marked resistance to Ca(2+)-induced disassembly. Polymers composed of normal alpha-tubulin and cleaved beta-tubulin or of cleaved alpha- and beta-tubulins were stabilized in the presence of added MAP2, myelin basic protein and histone H1. Cleavage of tubulin carboxy termini greatly potentiated microtubule stabilization by tau proteins. We show that this potentiation of polymer stabilization can be ascribed to tau-induced microtubule bundling. In our working conditions, such bundling upon association with tau proteins occurred only in the case of microtubules composed of cleaved alpha- and beta-tubulins and triggered apparent microtubule cross-stabilization among the bundled polymers. These results, as well as immunofluorescence analysis, which directly showed interactions between subtilisin-treated microtubules and MAPs, suggest that the carboxy termini of alpha- and beta-tubulins are not primarily involved in the binding of MAPs onto microtubules. However, interactions between tubulin carboxy termini and MAPs remain possible and might be involved in the regulation of MAP-induced microtubule bundling.

ABSTRACT Neuronal outgrowth occurs via coordinated remodeling of the cytoskeleton involving both actin and microtubules. Microtubule stabilization drives the extending neurite, yet little is known of the molecular mechanisms whereby extracellular cues regulate microtubule dynamics. Bone morphogenetic proteins (BMPs) play an important role in neuronal differentiation and morphogenesis, and BMP7 in particular induces the formation of dendrites. Here, we show that BMP7 induces stabilization of microtubules in both a MAP2-dependent neuronal cell culture model and in dendrites of primary cortical neurons. BMP7 rapidly activates c-Jun N-terminal kinases (JNKs), known regulators of microtubule dynamics, and we show that JNKs associate with the carboxy terminus of the BMP receptor, BMPRII. Activation and binding of JNKs to BMPRII is required for BMP7-induced microtubule stabilization and for BMP7-mediated dendrite formation in primary cortical neurons. These data indicate that BMPRII acts as a scaffold to localize and coordinate cytoskeletal remodeling and thereby provides an efficient means for extracellular cues, such as BMPs, to control neuronal dendritogenesis.

ABSTRACT The role of the herpes simplex virus type 1 tegument protein VP22 during infection is as yet undefined. We have previously shown that VP22 has the unusual property of efficient intercellular transport, such that the protein spreads from single expressing cells into large numbers of surrounding cells. We also noted that in cells expressing VP22 by transient transfection, the protein localizes in a distinctive cytoplasmic filamentous pattern. Here we show that this pattern represents a colocalization between VP22 and cellular microtubules. Moreover, we show that VP22 reorganizes microtubules into thick bundles which are easily distinguishable from nonbundled microtubules. These bundles are highly resistant to microtubule-depolymerizing agents such as nocodazole and incubation at 4°C, suggesting that VP22 has the capacity to stabilize the microtubule network. In addition, we show that the microtubules contained in these bundles are modified by acetylation, a marker for microtubule stability. Analysis of infected cells by both immunofluorescence and measurement of microtubule acetylation further showed that colocalization between VP22 and microtubules, and induction of microtubule acetylation, also occurs during infection. Taken together, these results suggest that VP22 exhibits the properties of a classical microtubule-associated protein (MAP) during both transfection and infection. This is the first demonstration of a MAP encoded by an animal virus.

In many eukaryotic cells going through M-phase, a bipolar spindle is formed by microtubules nucleated from centrosomes. These microtubules, in addition to being "captured" by kinetochores, may be stabilized by chromatin in two different ways: short-range stabilization effects may affect microtubules in close contact with the chromatin, while long-range stabilization effects may "guide" microtubule growth towards the chromatin (e.g., by introducing a diffusive gradient of an enzymatic activity that affects microtubule assembly). Here, we use both meiotic and mitotic extracts from Xenopus laevis eggs to study microtubule aster formation and microtubule dynamics in the presence of chromatin. In "low-speed" meiotic extracts, in the presence of salmon sperm chromatin, we find that short-range stabilization effects lead to a strong anisotropy of the microtubule asters. Analysis of the dynamic parameters of microtubule growth show that this anisotropy arises from a decrease in the catastrophe frequency, an increase in the rescue frequency and a decrease in the growth velocity. In this system we also find evidence for long-range "guidance" effects, which lead to a weak anisotropy of the asters. Statistically relevant results on these long-range effects are obtained in "high-speed" mitotic extracts in the presence of artificially constructed chromatin stripes. We find that aster anisotropy is biased in the direction of the chromatin and that the catastrophe frequency is reduced in its vicinity. In this system we also find a surprising dependence of the catastrophe and the rescue frequencies on the length of microtubules nucleated from centrosomes: the catastrophe frequency increase and the rescue frequency decreases with microtubule length.

La differenciation neuronale et les principales fonctions neuronales necessitent une forte stabilisation du cytosquelette microtubulaire. Les neurones contiennent une large proportion de microtubules qui resistent au froid et aux drogues depolymerisantes, et qui de plus possedent une dynamique fortement ralentie. L'origine de cette stabilisation n'est toujours pas clairement definie. Au cours de ce travail, nous avons etudie le role des proteines stop, une proteine regulee par la calmoduline, prealablement isolee a partir d'une population de microtubules stables au froid presente dans des cytosols de cerveaux de rats. Nous avons montre que dans les cellules neuronales, l'expression des proteines stop et des isoformes de proteines stop augmente au cours de la differenciation. Ces proteines sont associees a une large proportion de microtubules neuronaux et sont concentrees sur les microtubules stables au froid, resistants aux drogues et a longue duree de vie. L'inhibition des proteines stop abolit completement la stabilite au froid et aux drogues des microtubules presents dans les neurites preformes et empeche la formation de nouvelles extensions. Il apparait clairement que les proteines stop sont les principaux responsables du haut degre de stabilisation des microtubules observe dans les cellules neuronales et sont apparemment necessaires a la formation normale des extensions axodendritiques.

Die muskelspezifischen RING-Finger Ubiquitin E3 Ligasen MuRF1, MuRF2 und MuRF3 werden mit verschiedenen zellulären Prozessen in Verbindung gebracht. MuRF1 und MuRF3 beteiligen sich am Abbau mehrerer Muskelstrukturproteine über das Ubiquitin Proteasom System (UPS) und spielen somit eine wichtige Rolle bei der Aufrechterhaltung der Skelett- und Herzmuskelstruktur und -funktion. MuRF1 wurde als Atrophie-Marker identifiziert, da seine Expression während der Muskelatrophie ansteigt, und MuRF2 und MuRF3 wirken bei der Stabilisierung von Mikrotubuli und Differenzierung von Myozyten mit. Dennoch sind bisher viele Aspekte der Funktion von MuRF-Proteinen ungeklärt. Die Domänenstruktur der MuRF-Proteine zeigt mehrere hochkonservierte Domänen, die sich an Protein-Protein Interaktionen beteiligen. Die Identifizierung und Charakterisierung ihres Interaktoms ermöglicht ein besseres Verständnis ihrer Funktionen. Aus diesem Grund wurden quantitative massenspektrometrische Analysen durchgeführt, um neue Interaktionspartner und Substrate für MuRF1, 2 und 3 zu identifizieren. Sorting nexin 5 (SNX5), eine Untereinheit des Retromers in Säugetieren, wurde als Interaktionspartner von MuRF3 identifiziert. SNX5, das eine wichtige Rolle in subzellulären Transport-Signalwegen spielt, interagierte über seine BAR-Domäne mit MuRF3. SNX5 und MuRF3 co-lokalisierten und assoziierten mit vesikulären Strukturen des subzellulären Transport-Signalweges. SNX5 wurde außerdem als Substrat von MuRF2 identifiziert. MuRF2 band und ubiquitinierte SNX5 in vivo und vermittelte damit dessen Abbau über das UPS. MuRF3 stabilisierte SNX5 durch die Inhibierung dieses Abbaus. Somit konnten MuRF2 und MuRF3 mit einem in subzellulärem Transport aktiven Protein in Verbindung gebracht werden, das direkt mit Mikrotubuli assoziiert und funktionell von einem stabilen Mikrotubuli-Netzwerk abhängig ist. Dies legt eine mögliche regulatorische Rolle von MuRF2 und MuRF3 in Mikrotubuli-abhängigen subzellulären Transportwegen nahe.<br>Muscle specific RING-Finger ubiquitin E3 ligases MuRF1, MuRF2 and MuRF3 have been implicated in several cellular functions. MuRF1 and MuRF3 have been shown to bind and degrade muscle contractile and structural proteins via the ubiquitin proteasome system (UPS), thus playing an important role in the maintenance of skeletal and cardiac muscle structure and function. MuRF1 is considered an atrophy marker since its expression increases during muscle atrophy. MuRF2 and MuRF3 are involved in myocyte differentiation and both bind to and stabilize microtubules. Nevertheless, many aspects of the functions of the MuRF-family are unknown. The domain structure of the MuRF family implicates several highly conserved domains involved in protein-protein interaction. Accordingly, one way to better understand the role of MuRF proteins in myocyte function and protein homeostasis is to identify and characterize their interactome. Therefore, quantitative mass spectrometric analysis was used to identify novel interaction partners and target proteins of MuRF1, 2 and 3. Sorting nexin 5 (SNX5), a mammalian retromer subunit which plays an important role in subcellular trafficking pathways, was identified as a novel interaction partner of MuRF3, with which it interacted via its Bin/Amphiphysin/Rvs (BAR)-domain. SNX5 and MuRF3 co-localized and associated with early endosomes, connecting the microtubule-binding MuRF3 to structures of subcellular trafficking pathway. SNX5 was also identified as a substrate of MuRF2, which interacted with and ubiquitinated SNX5 in vivo, mediating its degradation in a UPS-dependent manner. This MuRF2-mediated degradation was inhibited by MuRF3, which stabilized SNX5. Thus, MuRF2 and MuRF3 were linked to a subcellular trafficking protein, SNX5, which is directly associated with microtubules and functionally dependent on a stable microtubule network, suggesting a possible regulatory role of MuRF2 and MuRF3 in microtubule-dependent subcellular trafficking pathways.

Microtubules are a fascinating component of the cellular scaffold protein network, the cytoskeleton. These hollow tubular structures are assembled of laterally associated proto-filaments containing ab-tubulin heterodimers in a head to tail arrangement. Accordingly microtubules have a defined polarity, which sets the base for the polarity of the cell. The microtubule lattice can be arranged in two conformations: In the more abundant B-lattice conformation, where the protofilaments interact laterally through a- to a- and b- to b-tubulin contacts and in the less stable A-lattice conformation, where a-tubulin interacts laterally with b-tubulin. In cells the microtubules generally contain 13 protofilaments of which usually one pair interacts in the A-lattice conformation, forming the so-called lattice seam. Microtubule dynamics and interactions are strongly regulated by micro-tubule associate proteins (MAPs). Structural investigations on MAPs and microtubule associated motor proteins in complex with microtubules have become possible in combination with modern electron microscopy (EM) and image processing. We have used biochemistry and different advanced EM techniques to study the interaction between microtubules and the MAP Mal3p in vitro. Mal3p is the sole member of the end-binding protein 1 (EB1) protein family in the fission yeast Schizosaccharomyces pombe. Previous in vivo studies have shown that Mal3p promotes microtubule growth. Our studies with high-resolution unidirectional shadowing EM revealed that Mal3p interacts with the microtubule lattice in a novel way, using binding sites on the microtubule that are different from those reported for other MAPs or motor proteins. Full-length Mal3p preferentially binds between two protofilaments on the microtubule lattice, leaving the rest of the lattice free. A case where Mal3p was found in two adjacent protofilament, revealed an A-lattice conformation on the microtubules, surprisingly indicating specific binding of Mal3p to the microtubule seam. With a lattice enhancer, in form of a b-tubulin binding kinesin motor domain, it was demonstrated that Mal3p stabilizes the seam which is thought to be the weakest part of a microtubule. Further, the presence of Mal3p during microtubule polymerization enhances the closure of protofilament sheets into a tubular organization. Cryo-EM and 3-D helical reconstruction on a monomeric microtubule binding domain of Mal3p, confirm the localization in between the protofilament and result in an accurate localization on the microtubule lattice. The results also indicate Mal3p’s capacity to influence the microtubule lattice conformation. Together, studies approached in vitro demonstrate that an EB1-family homolog not only interacts with the microtubule plus end, but also with the microtubule lattice. The structure of Mal3p interacting with microtubules reveals a new mechanism for microtubule stabilization and further insight on how plus end binding proteins are able promote microtubule growth. These findings further suggest that microtubules exhibit two distinct reaction platforms on their surface that can independently interact with selected MAPs or motors<br>Mikrotubuli sind eine faszinierende Komponente des Zytoskeletts einer Zelle. Ihre Struktur entspricht der eines Hohlzylinders. Sie sind aus seitlich assoziierten Proto-filamenten zusammengesetzt, die aus a- und b-Tubulin Untereinheiten bestehen. Diese Heterodimere sind gerichtet, bedingt durch ihre Kopf-Schwanz Anordnung. Folglich besitzen Mikrotubuli eine definierte Polarität, welche die Basis für die Polarität der Zelle bildet. Die Anordnung der Untereinheiten zu einem so genannten Mikrotubulus Gitter kann in zwei Konformationen vorkommen: In der häufigeren B-Gitter Formation, in welcher die Protofilamente seitlich durch a- zu a- und b- zu b-Tubulin interagieren und in der weniger stabilen A-Gitter Konformation, in der a-Tubulin lateral mit b-Tubulin wechselwirkt. In der Zelle vorkommende Mikrotubuli haben grundsätzlich 13 Proto-filamente. Mindestens ein Paar dieser Protofilamente interagiert in der A-Gitter Kon-formation und bildet die so genannte Gitter-Naht (lattice seam). Mikrotubuli Dynamik und Interaktionen sind streng durch Mikrotubuli assoziierte Proteine (MAPs) reguliert. Die Kombination aus moderner Elektronenmikroskopie (EM) und Bild-verarbeitung macht strukturelle Untersuchungen an MAPs und Motorproteinen im Zusammenhang mit Mikrutubuli möglich. Wir haben biochemische und hoch entwickelte EM Techniken benutzt, um die Interaktion zwischen Mikrotubuli und dem Mikrotubuli assoziierten Protein Mal3 in vitro zu untersuchen. Mal3p ist ein Homolog des konservierten Ende-Bindungs Protein 1 (EB1) in der Spalthefe Schizosaccharomyces pombe. Es wurde bereits gezeigt, dass EB1 die Struktur von Mikrotubuli stabilisiert. Mit Hilfe einer speziellen, hochauflösenden EM Schattierungstechnik haben wir demonstriert, dass Mal3p auf neuartige Weise mit dem Mikrotubulus Gitter interagiert. Dabei besetzt Mal3p Bindungsstellen am Mikrotubulus, die sich von denen der anderen MAPs oder Motorproteinen unterscheiden. Mal3p bevorzugt die Bindung zwischen zwei Proto-filamenten, lässt jedoch das übrigen Gitter unbesetzt. In seltenen Fällen wurde Mal3p in zwei nebeneinander angrenzenden Protofilamenten gefunden. An diesen Stellen zeigt sich überraschenderweise eine A-Gitter-Konformation am Mikrotubulus, was auf eine spezifische Naht-Bindung hinweist. Mit Hilfe einer Gitterverstärkung in Form einer Kinesin-Motor-Domäne, die an jede b-Untereinheit bindet, konnte gezeigt werden, dass Mal3p die Naht, den schwächsten Teil eines Mikrotubulus, stabilisiert. Des Weiteren unterstützt die Anwesenheit von Mal3p während der Mikrotubulus Polymerisation die Formierung zur Bildung des Hohlzylinders. Die Untersuchung der monomeren Mikrotubuli-Bindungs-Domäne von Mal3p unter Anwendung von Kryo-EM und anschließender 3-D helikalen Rekonstruktion, führte zur genauen Lokalisierung des Proteins auf dem Mikrotubulus Gerüst. Hierbei bestätigte sich auch die Lokalisation zwischen den Protofilamenten. Des Weiteren konnte gezeigt werden, dass Mal3p die Fähigkeit besitzt, die Konformation des Mikrotubulus Gitters zu beeinflussen. Zusammenfassend lässt sich sagen, dass das EB1-Homolog nicht nur an das Mikrotubulus Plus Ende, sondern auch an der Naht entlang des ganzen Mikrotubulus bindet. Die Art wie Mal3p mit den Mikrotubuli interagiert, zeigt einen neuen Mecha-nismus der Mikrotubuli Stabilisierung und eröffnet weitere Sichtweisen, wie Plus End Bindungsproteine die Dynamik von Mikrotubuli beeinflussen. Die Ergebnisse belegen, dass Mikrotubuli zwei definierte Reaktionsplattformen auf ihrer Oberfläche besitzen, die unabhängig mit verschiedenen MAPs und Motorproteinen interagieren

Cell-type specific dendritic morphologies emerge via complex growth mechanisms modulated by intrinsic and extrinsic signaling coupled with activity-dependent regulation. Combined, these processes converge on cytoskeletal effectors to direct dendritic arbor development, stabilize mature architecture, and facilitate structural plasticity. Transcription factors (TFs) function as essential cell intrinsic regulators of dendritogenesis involving both combinatorial and cell-type specific effects, however the molecular mechanisms via which these TFs govern arbor development and dynamics remain poorly understood. Studies in Drosophila dendritic arborization (da) sensory neurons have revealed combinatorial roles of the TFs Cut and Knot in modulating dendritic morphology, however putative convergent nodal points of Cut/Knot cytoskeletal regulation remain elusive. Here we use a combined neurogenomic, bioinformatic, and genetic approach to identify and molecularly characterize downstream effectors of these TFs. From these analyses, we identified Formin3 (Form3) as a convergent transcriptional target of both Cut and Knot. We demonstrate that Form3 functions cell-autonomously in class IV (CIV) da neurons to stabilize distal higher order branching along the proximal-distal axis of dendritic arbors. Furthermore, live confocal imaging of multi-fluor cytoskeletal reporters and IHC analyses reveal that form3 mutants exhibit a specific collapse of the dendritic microtubule (MT) cytoskeleton, the functional consequences of which include defective dendritic trafficking of mitochondria and satellite Golgi. Biochemical analyses reveal Form3 directly interacts with MTs via the FH1/FH2 domains. Form3 is predicted to interact with two alpha-tubulin N-acetyltransferases (ATAT1) suggesting it may promote MT stabilization via acetylation. Analyses of acetylated dendritic MTs supports this hypothesis as defects in form3 lead to reductions, whereas overexpression promotes increases in MT acetylation. Neurologically, mutations in Inverted Formin 2 (INF2; the human ortholog of form3) have been causally linked to dominant intermediate Charcot-Marie-Tooth (CMT) disease E. CMT sensory neuropathies lead to distal sensory loss resulting in a reduced ability to sense heat, cold, and pain. Intriguingly, disruption of form3 function in CIV nociceptive neurons results in a severe impairment in nocifensive behavior in response to noxious heat, which can be rescued by expression of INF2 revealing shared primordial functions in regulating nociception and providing novel mechanistic insights into the potential etiological bases of CMT sensory neuropathies.

The cytoskeleton consists primarily of microfilaments, microtubules, and intermediate filaments. Actin microfilaments have major role in growth, maintenance, and dynamic changes of growth cones and dendrites; stabilization of proteins at specific membrane locations; and vesicle dynamics during endocytosis and exocytosis. Microtubules provide the major tracks for intracellular transport and local cues for positioning of mitochondria and other organelles. The intermediate filaments in neurons are the neurofilaments that have a major role in regulating axonal caliber and mechanical stability. Glial fibrillary acid protein is a primary component of intermediate filaments in astrocytes. Nuclear lamins participate in regulation of the chromatin organization, trafficking of transcription factors across the nuclear envelope, and transduction of mechanical signals. Mutations affecting these cytoskeletal proteins produce a wide range of neurologic disorders, including neurodevelopmental disorders, peripheral neuropathies, myopathies, and leukodystrophy. All components of the cytoskeleton are involved in adult-onset neurodegenerative disorders.