scholarly journals Posttranslational Modifications of Tubulin and the Polarized Transport of Kinesin-1 in Neurons

2010 ◽  
Vol 21 (4) ◽  
pp. 572-583 ◽  
Author(s):  
Jennetta W. Hammond ◽  
Chun-Fang Huang ◽  
Stefanie Kaech ◽  
Catherine Jacobson ◽  
Gary Banker ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Hippocampal Neurons ◽  
Neuronal Development ◽  
Glycogen Synthase ◽  
Morphological Differentiation ◽  
Tubulin Acetylation ◽  
Microtubule Stability ◽  
Polarized Transport ◽  
And Function ◽  
Selective Accumulation

Polarized transport by microtubule-based motors is critical for neuronal development and function. Selective translocation of the Kinesin-1 motor domain is the earliest known marker of axonal identity, occurring before morphological differentiation. Thus, Kinesin-1–mediated transport may contribute to axonal specification. We tested whether posttranslational modifications of tubulin influence the ability of Kinesin-1 motors to distinguish microtubule tracks during neuronal development. We detected no difference in microtubule stability between axons and minor neurites in polarized stage 3 hippocampal neurons. In contrast, microtubule modifications were enriched in a subset of neurites in unpolarized stage 2 cells and the developing axon in polarized stage 3 cells. This enrichment correlated with the selective accumulation of constitutively active Kinesin-1 motors. Increasing tubulin acetylation, without altering the levels of other tubulin modifications, did not alter the selectivity of Kinesin-1 accumulation in polarized cells. However, globally enhancing tubulin acetylation, detyrosination, and polyglutamylation by Taxol treatment or inhibition of glycogen synthase kinase 3β decreased the selectivity of Kinesin-1 translocation and led to the formation of multiple axons. Although microtubule acetylation enhances the motility of Kinesin-1, the preferential translocation of Kinesin-1 on axonal microtubules in polarized neuronal cells is not determined by acetylation alone but is probably specified by a combination of tubulin modifications.

scholarly journals Microtubule stabilization specifies initial neuronal polarization

2008 ◽  
Vol 180 (3) ◽  
pp. 619-632 ◽  
Author(s):  
Harald Witte ◽  
Dorothee Neukirchen ◽  
Frank Bradke
Keyword(s):  
Hippocampal Neurons ◽  
Glycogen Synthase ◽  
Initial Step ◽  
Microtubule Dynamics ◽  
Neuronal Polarity ◽  
Low Doses ◽  
Microtubule Stabilization ◽  
Physiological Signal ◽  
Microtubule Stability ◽  
Neuronal Polarization

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.

scholarly journals Cofilin and Actin Dynamics: Multiple Modes of Regulation and Their Impacts in Neuronal Development and Degeneration

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2726
Author(s):  
James R. Bamburg ◽  
Laurie S. Minamide ◽  
O’Neil Wiggan ◽  
Lubna H. Tahtamouni ◽  
Thomas B. Kuhn
Keyword(s):  
Cortical Neurons ◽  
Hippocampal Neurons ◽  
Translational Regulation ◽  
Neuronal Development ◽  
Mitochondrial Fission ◽  
Amyloid Β ◽  
Cellular Prion Protein ◽  
Actin Dynamics ◽  
Specific Regulation ◽  
And Function

Proteins of the actin depolymerizing factor (ADF)/cofilin family are ubiquitous among eukaryotes and are essential regulators of actin dynamics and function. Mammalian neurons express cofilin-1 as the major isoform, but ADF and cofilin-2 are also expressed. All isoforms bind preferentially and cooperatively along ADP-subunits in F-actin, affecting the filament helical rotation, and when either alone or when enhanced by other proteins, promotes filament severing and subunit turnover. Although self-regulating cofilin-mediated actin dynamics can drive motility without post-translational regulation, cells utilize many mechanisms to locally control cofilin, including cooperation/competition with other proteins. Newly identified post-translational modifications function with or are independent from the well-established phosphorylation of serine 3 and provide unexplored avenues for isoform specific regulation. Cofilin modulates actin transport and function in the nucleus as well as actin organization associated with mitochondrial fission and mitophagy. Under neuronal stress conditions, cofilin-saturated F-actin fragments can undergo oxidative cross-linking and bundle together to form cofilin-actin rods. Rods form in abundance within neurons around brain ischemic lesions and can be rapidly induced in neurites of most hippocampal and cortical neurons through energy depletion or glutamate-induced excitotoxicity. In ~20% of rodent hippocampal neurons, rods form more slowly in a receptor-mediated process triggered by factors intimately connected to disease-related dementias, e.g., amyloid-β in Alzheimer’s disease. This rod-inducing pathway requires a cellular prion protein, NADPH oxidase, and G-protein coupled receptors, e.g., CXCR4 and CCR5. Here, we will review many aspects of cofilin regulation and its contribution to synaptic loss and pathology of neurodegenerative diseases.

scholarly journals Effects of α-tubulin acetylation on microtubule structure and stability

2019 ◽  
Vol 116 (21) ◽  
pp. 10366-10371 ◽  
Author(s):  
Lisa Eshun-Wilson ◽  
Rui Zhang ◽  
Didier Portran ◽  
Maxence V. Nachury ◽  
Daniel B. Toso ◽  
...  
Keyword(s):  
Conformational Sampling ◽  
Conformational Ensembles ◽  
Microtubule Stabilization ◽  
Flexible Loop ◽  
Tubulin Acetylation ◽  
Microtubule Stability ◽  
Cryo Electron Microscopy ◽  
Conformational Landscape ◽  
Dynamics Simulations ◽  
And Function

Acetylation of K40 in α-tubulin is the sole posttranslational modification to mark the luminal surface of microtubules. It is still controversial whether its relationship with microtubule stabilization is correlative or causative. We have obtained high-resolution cryo-electron microscopy (cryo-EM) reconstructions of pure samples of αTAT1-acetylated and SIRT2-deacetylated microtubules to visualize the structural consequences of this modification and reveal its potential for influencing the larger assembly properties of microtubules. We modeled the conformational ensembles of the unmodified and acetylated states by using the experimental cryo-EM density as a structural restraint in molecular dynamics simulations. We found that acetylation alters the conformational landscape of the flexible loop that contains αK40. Modification of αK40 reduces the disorder of the loop and restricts the states that it samples. We propose that the change in conformational sampling that we describe, at a location very close to the lateral contacts site, is likely to affect microtubule stability and function.

scholarly journals Targeted polyubiquitylation of RASSF1C by the Mule and SCFβ-TrCP ligases in response to DNA damage

2011 ◽  
Vol 441 (1) ◽  
pp. 227-236 ◽  
Author(s):  
Xin Zhou ◽  
Ting-Ting Li ◽  
Xu Feng ◽  
Esther Hsiang ◽  
Yue Xiong ◽  
...  
Keyword(s):  
Dna Damage ◽  
Uv Irradiation ◽  
Glycogen Synthase ◽  
E3 Ligase ◽  
Cycle Arrest ◽  
Microtubule Stability ◽  
C Terminus ◽  
Stress Signals ◽  
Phosphoinositide 3 Kinase ◽  
And Function

RASSF1A [Ras association (RalGDS/AF-6) domain family member 1A] and RASSF1C are two ubiquitously expressed isoforms of the RASSF1 gene. The promoter of RASSF1A is frequently hypermethylated, resulting in inactivation in various human cancers. RASSF1A is implicated in the regulation of apoptosis, microtubule stability and cell cycle arrest. However, little is known about the regulation and function of RASSF1C. In the present study we show that exogenously expressed RASSF1C is a very unstable protein that is highly polyubiquitylated and degraded via the proteasome. Furthermore, RASSF1C degradation is enhanced when cells are exposed to stress signals, such as UV irradiation. Mule, a HECT (homologous with E6-associated protein C-terminus) family E3 ligase, but not SCFβ-TrCP [where SCF is Skp1 (S-phase kinase-associated protein 1)/cullin/F-box and β-TrCP is β-bransducin repeat-containing protein] or CUL4 (cullin 4)-DDB1 (damage-specific DNA-binding protein 1), is the E3 ligase for RASSF1C under normal conditions, whereas both Mule and SCFβ-TrCP target RASSF1C degradation in response to UV irradiation. GSK3 (glycogen synthase kinase 3) phosphorylates RASSF1C to promote RASSF1C degradation subsequently, which is negatively regulated by the PI3K (phosphoinositide 3-kinase)/Akt pathway. Thus the present study reveals a novel regulation of RASSF1C and the potentially important role of RASSF1C in DNA damage responses.

scholarly journals Effects of α-tubulin acetylation on microtubule structure and stability

2019 ◽  
Author(s):  
Lisa Eshun-Wilson ◽  
Rui Zhang ◽  
Didier Portran ◽  
Maxence Nachury ◽  
Dan Toso ◽  
...  
Keyword(s):  
Conformational Sampling ◽  
Post Translational Modification ◽  
Conformational Ensembles ◽  
Microtubule Stabilization ◽  
Tubulin Acetylation ◽  
Microtubule Stability ◽  
Cryo Electron Microscopy ◽  
Conformational Landscape ◽  
Dynamics Simulations ◽  
And Function

ABSTRACTAcetylation of K40 in α-tubulin is the sole post-translational modification to mark the luminal surface of microtubules. It is still controversial whether its relationship with microtubule stabilization is correlative or causative. We have obtained high-resolution cryo-electron microscopy reconstructions of pure samples of αTAT1-acetylated and SIRT2-deacetylated microtubules to visualize the structural consequences of this modification and reveal its potential for influencing the larger assembly properties of microtubules. We modeled the conformational ensembles of the unmodified and acetylated states by using the experimental cryo-EM density as the structural restraint in molecular dynamics simulations. We found that acetylation alters the conformational landscape of the flexible loop that contains αK40. Modification of αK40 reduces the disorder of the loop and restricts the states that it samples. We propose that the change in conformational sampling that we describe, at a location very close to the lateral contacts site, is likely to affect microtubule stability and function.

scholarly journals Sarm1, a negative regulator of innate immunity, interacts with syndecan-2 and regulates neuronal morphology

2011 ◽  
Vol 193 (4) ◽  
pp. 769-784 ◽  
Author(s):  
Chiung-Ya Chen ◽  
Chia-Wen Lin ◽  
Chiung-Ying Chang ◽  
Si-Tse Jiang ◽  
Yi-Ping Hsueh
Keyword(s):  
Innate Immunity ◽  
Hippocampal Neurons ◽  
Dendritic Arborization ◽  
Neuronal Morphology ◽  
Multiple Roles ◽  
Negative Regulator ◽  
Jnk Pathway ◽  
Neuronal Morphogenesis ◽  
Tubulin Acetylation ◽  
Microtubule Stability

Dendritic arborization is a critical neuronal differentiation process. Here, we demonstrate that syndecan-2 (Sdc2), a synaptic heparan sulfate proteoglycan that triggers dendritic filopodia and spine formation, regulates dendritic arborization in cultured hippocampal neurons. This process is controlled by sterile α and TIR motif–containing 1 protein (Sarm1), a negative regulator of Toll-like receptor 3 (TLR3) in innate immunity signaling. We show that Sarm1 interacts with and receives signal from Sdc2 and controls dendritic arborization through the MKK4–JNK pathway. In Sarm1 knockdown mice, dendritic arbors of neurons were less complex than those of wild-type littermates. In addition to acting downstream of Sdc2, Sarm1 is expressed earlier than Sdc2, which suggests that it has multiple roles in neuronal morphogenesis. Specifically, it is required for proper initiation and elongation of dendrites, axonal outgrowth, and neuronal polarization. These functions likely involve Sarm1-mediated regulation of microtubule stability, as Sarm1 influenced tubulin acetylation. This study thus reveals the molecular mechanism underlying the action of Sarm1 in neuronal morphogenesis.

scholarly journals CAMSAP3 maintains neuronal polarity through regulation of microtubule stability

2018 ◽  
Vol 115 (39) ◽  
pp. 9750-9755 ◽  
Author(s):  
Varisa Pongrakhananon ◽  
Hiroko Saito ◽  
Sylvain Hiver ◽  
Takaya Abe ◽  
Go Shioi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Neuronal Polarity ◽  
Wild Type ◽  
Tubulin Acetylation ◽  
Microtubule Stability ◽  
Single Axon ◽  
Key Enzyme

The molecular mechanisms that guide each neuron to become polarized, forming a single axon and multiple dendrites, remain unknown. Here we show that CAMSAP3 (calmodulin-regulated spectrin-associated protein 3), a protein that regulates the minus-end dynamics of microtubules, plays a key role in maintaining neuronal polarity. In mouse hippocampal neurons, CAMSAP3 was enriched in axons. Although axonal microtubules were generally acetylated, CAMSAP3 was preferentially localized along a less-acetylated fraction of the microtubules. CAMSAP3-mutated neurons often exhibited supernumerary axons, along with an increased number of neurites having nocodazole-resistant/acetylated microtubules compared with wild-type neurons. Analysis using cell lines showed that CAMSAP3 depletion promoted tubulin acetylation, and conversely, mild overexpression of CAMSAP3 inhibited it, suggesting that CAMSAP3 works to retain nonacetylated microtubules. In contrast, CAMSAP2, a protein related to CAMSAP3, was detected along all neurites, and its loss did not affect neuronal polarity, nor did it cause increased tubulin acetylation. Depletion of α-tubulin acetyltransferase-1 (αTAT1), the key enzyme for tubulin acetylation, abolished CAMSAP3 loss-dependent multiple-axon formation. These observations suggest that CAMSAP3 sustains a nonacetylated pool of microtubules in axons, interfering with the action of αTAT1, and this process is important to maintain neuronal polarity.

Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications

2020 ◽  
Vol 19 ◽  
Author(s):  
Sumei Li ◽  
Jifeng Zhang ◽  
Jiaqi Zhang ◽  
Jiong Li ◽  
Longfei Cheng ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Hippocampal Neurons ◽  
Neuronal Development ◽  
Phosphorylation Site ◽  
Microtubule Dynamics ◽  
Microtubule Assembly ◽  
C Terminus ◽  
Mediator Protein ◽  
Branch Formation

Aims: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development, and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury. Background: Microtubule polymerization and severing are the basis for the neurite outgrowth and branch formation. Collapsin response mediator protein 2 (CRMP2) regulates axonal growth and branching as a binding partner of the tubulin heterodimer to promote microtubule assembly. And spastin participates in the growth and regeneration of neurites by severing microtubules into small segments. However, how CRMP2 and spastin cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. Objective: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin. Method: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites. Result: We first demonstrated that CRMP2 interacted with spastin to promote the neurite outgrowth and branch formation. Furthermore, our results identified that phosphorylation modification failed to alter the binding affinities of CRMP2 for spastin, but inhibited their binding to microtubules. CRMP2 interacted with the MTBD domain of spastin via its C-terminus, and blocking the binding sites of them inhibited the outgrowth and branch formation of neurites. In addition, we confirmed one phosphorylation site S210 at spastin in hippocampal neurons and phosphorylation spastin at site S210 promoted the neurite outgrowth but not branch formation by remodeling microtubules. Conclusion: Taken together, our data demonstrated that the interaction of CRMP2 and spastin is required for neurite outgrowth and branch formation and their interaction is not regulated by their phosphorylation.

scholarly journals PKC-phosphorylation of Liprin-α3 triggers phase separation and controls presynaptic active zone structure

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Javier Emperador-Melero ◽  
Man Yan Wong ◽  
Shan Shan H. Wang ◽  
Giovanni de Nola ◽  
Hajnalka Nyitrai ◽  
...  
Keyword(s):  
Phase Separation ◽  
Active Zone ◽  
Neurotransmitter Release ◽  
Hippocampal Neurons ◽  
Phosphorylation Site ◽  
Double Knockout ◽  
Zone Structure ◽  
Presynaptic Nerve Terminal ◽  
Condensate Formation ◽  
And Function

AbstractThe active zone of a presynaptic nerve terminal defines sites for neurotransmitter release. Its protein machinery may be organized through liquid–liquid phase separation, a mechanism for the formation of membrane-less subcellular compartments. Here, we show that the active zone protein Liprin-α3 rapidly and reversibly undergoes phase separation in transfected HEK293T cells. Condensate formation is triggered by Liprin-α3 PKC-phosphorylation at serine-760, and RIM and Munc13 are co-recruited into membrane-attached condensates. Phospho-specific antibodies establish phosphorylation of Liprin-α3 serine-760 in transfected cells and mouse brain tissue. In primary hippocampal neurons of newly generated Liprin-α2/α3 double knockout mice, synaptic levels of RIM and Munc13 are reduced and the pool of releasable vesicles is decreased. Re-expression of Liprin-α3 restored these presynaptic defects, while mutating the Liprin-α3 phosphorylation site to abolish phase condensation prevented this rescue. Finally, PKC activation in these neurons acutely increased RIM, Munc13 and neurotransmitter release, which depended on the presence of phosphorylatable Liprin-α3. Our findings indicate that PKC-mediated phosphorylation of Liprin-α3 triggers its phase separation and modulates active zone structure and function.

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