scholarly journals Desmin intermediate filaments and tubulin detyrosination stabilize growing microtubules in the cardiomyocyte

2021 ◽  
Author(s):  
Alexander Salomon ◽  
Naima Okami ◽  
Julie Heffler ◽  
Jia-Jye Lee ◽  
Patrick Robison ◽  
...  
Keyword(s):  
Intermediate Filaments ◽  
Muscle Mechanics ◽  
High Energy ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Microtubule Network ◽  
Biochemical Assays ◽  
Molecular Determinants ◽  
Microtubule Growth ◽  
Dynamic Microtubule

The microtubule network of the cardiomyocyte exhibits specialized architecture, stability and mechanical behavior that accommodate the demands of working muscle cells. Stable, post-translationally detyrosinated microtubules are physical coupled to the sarcomere, the contractile apparatus of muscle, and resist sarcomere motion to regulate muscle mechanics and mechanosignaling. Control of microtubule growth and shrinkage dynamics represents a potential intermediate in the formation of a stable, physically coupled microtubule network, yet the molecular determinants that govern dynamics are unknown. Here we test the hypothesis that desmin intermediate filaments may stabilize growing microtubules at the sarcomere Z-disk in a detyrosination-dependent manner. Using a combination of biochemical assays and direct observation of microtubule plus-end dynamics in primary adult cardiomyocytes, we determine that: 1) tyrosination increases the frequency of microtubule depolymerization and reduces the pausing of microtubules at the Z-disk, leading to a more dynamic microtubule; and 2) desmin intermediate filaments stabilize both growing and shrinking microtubules specifically at the Z-disk and protect them from depolymerization. This stabilizes iteratively growing, detyrosinated microtubules between adjacent sarcomeres, which promotes the formation of high-energy microtubules that buckle between sarcomeres and elevates myocyte viscoelasticity. Our findings inform on how the tubulin code and intermediate filaments regulate microtubule dynamics, and provide mechanism to the establishment of a spatially organized, physically coupled, and long-lived microtubule network in the cardiomyocyte.

scholarly journals KIF2C regulates synaptic plasticity and cognition by mediating dynamic microtubule invasion of dendritic spines

2021 ◽  
Author(s):  
Rui Zheng ◽  
Yonglan Du ◽  
Xintai Wang ◽  
Tailin Liao ◽  
Zhe Zhang ◽  
...  
Keyword(s):  
Nervous System ◽  
Synaptic Plasticity ◽  
Cell Structure ◽  
Critical Role ◽  
Long Term Potentiation ◽  
Conditional Knockout ◽  
Synaptic Membrane ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Dynamic Microtubule

Dynamic microtubules play a critical role in cell structure and function. In nervous system, microtubules specially extend into and out of synapses to regulate synaptic development and plasticity. However, the detailed polymerization especially the depolymerization mechanism that regulates dynamic microtubules in synapses is still unclear. In this study, we find that KIF2C, a dynamic microtubule depolymerization protein without known function in the nervous system, plays a vital role in the structural and functional plasticity of synapses and regulates cognitive function. Using RNAi knockdown and conditional knockout approaches, we showed that KIF2C regulates spine morphology and synaptic membrane expression of AMPA (α- amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) receptors. Moreover, KIF2C deficiency leads to impaired excitatory transmission, long-term potentiation, and altered cognitive behaviors in mice. Mechanistically, KIF2C regulates microtubule dynamics and microtubule invasion of spines in neurons by its microtubule depolymerization capability in a neuronal activity-dependent manner. This study explores a novel function of KIF2C in the nervous system and provides an important regulatory mechanism on how microtubule invasion of spines regulates synaptic plasticity and cognition behaviors.

scholarly journals Mitotic spindle disassembly in human cells relies on CRIPT-bearing hierarchical redox signals

2019 ◽  
Author(s):  
Kehan Xu ◽  
Lingling Yang ◽  
Xiu Cheng ◽  
Xiaoyan Liu ◽  
Hao Huang ◽  
...  
Keyword(s):  
Human Cells ◽  
Live Cell ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Cysteine Substitution ◽  
Cysteine Rich Protein ◽  
Biochemical Assays ◽  
Spindle Disassembly ◽  
Live Cell Microscopy ◽  
Cell Microscopy

AbstractSwift and complete spindle disassembly is essential for cell survival, yet how it happens is largely unknown. Here we used real-time live-cell microscopy and biochemical assays to show that a cysteine-rich protein CRIPT dictates the spindle disassembly in a redox-dependent manner in human cells. This previously reported cytoplasmic protein was found to have a confined nuclear localization during interphase but was distributed to spindles and underwent redox modifications to form disulfides within CXXC pairs during mitosis. Then, it interacts with and transfers redox response to tubulin subunits to induce microtubule depolymerization. The mutants with any of cysteine substitution completely block the spindle disassembly generating two cell populations with long-lasting metaphase spindles or spindle remnants. The live cell recordings of a disease-relevant mutant (CRIPTC3Y) revealed that microtubule depolymerization at spindle ends during anaphase and the entire spindle dissolution during telophase may share a common CRIPT-bearing redox-controlled mechanism.

The stathmin phosphoprotein family: intracellular localization and effects on the microtubule network

1998 ◽  
Vol 111 (22) ◽  
pp. 3333-3346 ◽  
Author(s):  
O. Gavet ◽  
S. Ozon ◽  
V. Manceau ◽  
S. Lawler ◽  
P. Curmi ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Intracellular Signaling ◽  
Splice Variants ◽  
Intracellular Localization ◽  
Phosphorylation Site ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Microtubule Network

Stathmin is a small regulatory phosphoprotein integrating diverse intracellular signaling pathways. It is also the generic element of a protein family including the neural proteins SCG10, SCLIP, RB3 and its two splice variants RB3′ and RB3″. Stathmin itself was shown to interact in vitro with tubulin in a phosphorylation-dependent manner, sequestering free tubulin and hence promoting microtubule depolymerization. We investigated the intracellular distribution and tubulin depolymerizing activity in vivo of all known members of the stathmin family. Whereas stathmin is not associated with interphase microtubules in HeLa cells, a fraction of it is concentrated at the mitotic spindle. We generated antisera specific for stathmin phosphoforms, which allowed us to visualize the regulation of phosphorylation-dephosphorylation during the successive stages of mitosis, and the partial localization of stathmin phosphorylated on serine 16 at the mitotic spindle. Results from overexpression experiments of wild-type and novel phosphorylation site mutants of stathmin further suggest that it induces depolymerization of interphase and mitotic microtubules in its unphosphorylated state but is inactivated by phosphorylation in mitosis. Phosphorylation of mutants 16A25A and 38A63A on sites 38 and 63 or 16 and 25, respectively, was sufficient for the formation of a functional spindle, whereas mutant 16A25A38A63E retained a microtubule depolymerizing activity. Transient expression of each of the neural phosphoproteins of the stathmin family showed that they are at least partially associated to the Golgi apparatus and not to other major membrane compartments, probably through their different NH2-terminal domains, as described for SCG10. Most importantly, like stathmin and SCG10, overexpressed SCLIP, RB3 and RB3″ were able to depolymerize interphase microtubules. Altogether, our results demonstrate in vivo the functional conservation of the stathmin domain within each protein of the stathmin family, with a microtubule destabilizing activity most likely essential for their specific biological function(s).

scholarly journals A liquid +TIP-network drives microtubule dynamics through tubulin condensation

2021 ◽  
Author(s):  
Julie Miesch ◽  
Robert T. Wimbish ◽  
Marie-Claire Velluz ◽  
Charlotte Aumeier
Keyword(s):  
Phase Separation ◽  
Binding Proteins ◽  
Tip Growth ◽  
Regulatory Mechanism ◽  
Microtubule Dynamics ◽  
Microtubule Network ◽  
Cell Behaviors ◽  
Dynamic Cell ◽  
Microtubule Growth ◽  
Dynamic Microtubule

Tubulin dimers assemble into a dynamic microtubule network throughout the cell. Microtubule dynamics and network organization must be precisely tuned for the microtubule cytoskeleton to regulate a dazzling array of dynamic cell behaviors. Since tubulin concentration determines microtubule growth, we studied here a novel regulatory mechanism of microtubule dynamics: local tubulin condensation. We discovered that two microtubule tip-binding proteins, CLIP-170 and EB3, undergo phase separation and form an EB3/CLIP-170 droplet at the growing microtubule tip. We prove that this +TIP-droplet has the capacity to locally condense tubulin. This process of tubulin co-condensation is spatially initiated at the microtubule tip and temporally regulated to occur only when there is tip growth. Tubulin condensation at the growing microtubule tip increases growth speeds three-fold and strongly reduces depolymerization events. With this work we establish a new mechanism to regulate microtubule dynamics by enrichment of tubulin at strategically important locations: the growing microtubule tips.

scholarly journals Sigma-1 Receptor Promotes Mitochondrial Bioenergetics by Orchestrating ER Ca2+ Leak during Early ER Stress

Metabolites ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 422
Author(s):  
Zhanat Koshenov ◽  
Furkan E. Oflaz ◽  
Martin Hirtl ◽  
Johannes Pilic ◽  
Olaf A. Bachkoenig ◽  
...  
Keyword(s):  
Er Stress ◽  
Energy Demand ◽  
Molecular Mechanisms ◽  
High Energy ◽  
Human Neuroblastoma Cell ◽  
Mitochondrial Bioenergetics ◽  
Dependent Manner ◽  
Human Neuroblastoma ◽  
Sigma 1 Receptor ◽  
Sigma 1

The endoplasmic reticulum (ER) is a complex, multifunctional organelle of eukaryotic cells and responsible for the trafficking and processing of nearly 30% of all human proteins. Any disturbance to these processes can cause ER stress, which initiates an adaptive mechanism called unfolded protein response (UPR) to restore ER functions and homeostasis. Mitochondrial ATP production is necessary to meet the high energy demand of the UPR, while the molecular mechanisms of ER to mitochondria crosstalk under such stress conditions remain mainly enigmatic. Thus, better understanding the regulation of mitochondrial bioenergetics during ER stress is essential to combat many pathologies involving ER stress, the UPR, and mitochondria. This article investigates the role of Sigma-1 Receptor (S1R), an ER chaperone, has in enhancing mitochondrial bioenergetics during early ER stress using human neuroblastoma cell lines. Our results show that inducing ER stress with tunicamycin, a known ER stressor, greatly enhances mitochondrial bioenergetics in a time- and S1R-dependent manner. This is achieved by enhanced ER Ca2+ leak directed towards mitochondria by S1R during the early phase of ER stress. Our data point to the importance of S1R in promoting mitochondrial bioenergetics and maintaining balanced H2O2 metabolism during early ER stress.

scholarly journals Microtubule-dependent distribution of mRNA in adult cardiocytes

2008 ◽  
Vol 294 (3) ◽  
pp. H1135-H1144 ◽  
Author(s):  
Dimitri Scholz ◽  
Catalin F. Baicu ◽  
William J. Tuxworth ◽  
Lin Xu ◽  
Harinath Kasiganesan ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Fluorescent Protein ◽  
Average Distance ◽  
Yellow Fluorescent Protein ◽  
Pressure Overload ◽  
Structural Protein ◽  
Protein Fusion ◽  
Microtubule Depolymerization ◽  
Microtubule Network ◽  
Hypertrophic Growth

Synthesis of myofibrillar proteins in the diffusion-restricted adult cardiocyte requires microtubule-based active transport of mRNAs as part of messenger ribonucleoprotein particles (mRNPs) to translation sites adjacent to nascent myofibrils. This is especially important for compensatory hypertrophy in response to hemodynamic overloading. The hypothesis tested here is that excessive microtubule decoration by microtubule-associated protein 4 (MAP4) after cardiac pressure overloading could disrupt mRNP transport and thus hypertrophic growth. MAP4-overexpressing and pressure-overload hypertrophied adult feline cardiocytes were infected with an adenovirus encoding zipcode-binding protein 1-enhanced yellow fluorescent protein fusion protein, which is incorporated into mRNPs, to allow imaging of these particles. Speed and distance of particle movement were measured via time-lapse microscopy. Microtubule depolymerization was used to study microtubule-based transport and distribution of mRNPs. Protein synthesis was assessed as radioautographic incorporation of [3H]phenylalanine. After microtubule depolymerization, mRNPs persist only perinuclearly and apparent mRNP production and protein synthesis decrease. Reestablishing microtubules restores mRNP production and transport as well as protein synthesis. MAP4 overdecoration of microtubules via adenovirus infection in vitro or following pressure overloading in vivo reduces the speed and average distance of mRNP movement. Thus cardiocyte microtubules are required for mRNP transport and structural protein synthesis, and MAP4 decoration of microtubules, whether directly imposed or accompanying pressure-overload hypertrophy, causes disruption of mRNP transport and protein synthesis. The dense, highly MAP4-decorated microtubule network seen in severe pressure-overload hypertrophy both may cause contractile dysfunction and, perhaps even more importantly, may prevent a fully compensatory growth response to hemodynamic overloading.

scholarly journals The EF-Hand Ca2+-binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca2+-binding Requirements

2004 ◽  
Vol 15 (2) ◽  
pp. 481-496 ◽  
Author(s):  
Josefa Andrade ◽  
Hu Zhao ◽  
Brian Titus ◽  
Sandra Timm Pearce ◽  
Margarida Barroso
Keyword(s):  
Endoplasmic Reticulum ◽  
Conformational Changes ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Network Formation ◽  
Binding Protein ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Independent Manner ◽  
Ef Hand

We have reported that p22, an N-myristoylated EF-hand Ca2+-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca2+, p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca2+-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based “bulk microinjection” assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca2+-binding p22 mutant, which is unable to undergo Ca2+-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca2+-independent manner and affects ER network assembly in a Ca2+-dependent manner.

scholarly journals Specificity and mechanism of carbohydrate demethylation by cytochrome P450 monooxygenases

2018 ◽  
Vol 475 (23) ◽  
pp. 3875-3886 ◽  
Author(s):  
Craig S. Robb ◽  
Lukas Reisky ◽  
Uwe T. Bornscheuer ◽  
Jan-Hendrik Hehemann
Keyword(s):  
Cytochrome P450 ◽  
Active Site ◽  
Substrate Recognition ◽  
High Energy ◽  
Cytochrome P450 Monooxygenases ◽  
P450 Monooxygenases ◽  
High Energy Barrier ◽  
Molecular Determinants ◽  
Cytochrome P450 Family ◽  
Carbohydrate Ligand

Degradation of carbohydrates by bacteria represents a key step in energy metabolism that can be inhibited by methylated sugars. Removal of methyl groups, which is critical for further processing, poses a biocatalytic challenge because enzymes need to overcome a high energy barrier. Our structural and computational analysis revealed how a member of the cytochrome P450 family evolved to oxidize a carbohydrate ligand. Using structural biology, we ascertained the molecular determinants of substrate specificity and revealed a highly specialized active site complementary to the substrate chemistry. Invariance of the residues involved in substrate recognition across the subfamily suggests that they are critical for enzyme function and when mutated, the enzyme lost substrate recognition. The structure of a carbohydrate-active P450 adds mechanistic insight into monooxygenase action on a methylated monosaccharide and reveals the broad conservation of the active site machinery across the subfamily.

The Kar3p and Kip2p motors function antagonistically at the spindle poles to influence cytoplasmic microtubule numbers

1998 ◽  
Vol 111 (3) ◽  
pp. 295-301 ◽  
Author(s):  
A. Huyett ◽  
J. Kahana ◽  
P. Silver ◽  
X. Zeng ◽  
W.S. Saunders
Keyword(s):  
Molecular Motors ◽  
Motor Proteins ◽  
Single Gene ◽  
Intermediate Phenotype ◽  
Microtubule Network ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cytoplasmic Microtubule ◽  
Spindle Poles ◽  
Microtubule Motors ◽  
Microtubule Growth

Microtubules provide the substrate for intracellular trafficking by association with molecular motors of the kinesin and dynein superfamilies. Motor proteins are generally thought to function as force generating units for transport of various cargoes along the microtubule polymer. Recent work suggests additional roles for motor proteins in changing the structure of the microtubule network itself. We report here that in the budding yeast Saccharomyces cerevisiae microtubule motors have antagonistic effects on microtubule numbers and lengths. As shown previously, loss of the Kar3p motor stimulates cytoplasmic microtubule growth while loss of Kip2p leads to a sharp reduction in cytoplasmic microtubule numbers. Loss of both the Kip2p and Kar3p motors together in the same cell produces an intermediate phenotype, suggesting that these two motors act in opposition to control cytoplasmic microtubule density. A Kip2p-GFP fusion from single gene expression is most concentrated at the spindle poles, as shown previously for an epitope tagged Kar3p-HA, suggesting both of these motors act from the minus ends of the microtubules to influence microtubule numbers.

Centrosome cohesion is regulated by a balance of kinase and phosphatase activities

2001 ◽  
Vol 114 (20) ◽  
pp. 3749-3757 ◽  
Author(s):  
Patrick Meraldi ◽  
Erich A. Nigg
Keyword(s):  
Protein Phosphatase ◽  
Kinase Activity ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Microtubule Depolymerization ◽  
Drug Induced ◽  
Microtubule Network ◽  
Similar Phenotype ◽  
Underlying Mechanisms

Centrosome cohesion and separation are regulated throughout the cell cycle, but the underlying mechanisms are not well understood. Since overexpression of a protein kinase, Nek2, is able to trigger centrosome splitting (the separation of parental centrioles), we have surveyed a panel of centrosome-associated kinases for their ability to induce a similar phenotype. Cdk2, in association with either cyclin A or E, was as effective as Nek2, but several other kinases tested did not significantly interfere with centrosome cohesion. Centrosome splitting could also be triggered by inhibition of phosphatases, and protein phosphatase 1α (PP1α) was identified as a likely physiological antagonist of Nek2. Furthermore, we have revisited the role of the microtubule network in the control of centrosome cohesion. We could confirm that microtubule depolymerization by nocodazole causes centrosome splitting. Surprisingly, however, this drug-induced splitting also required kinase activity and could specifically be suppressed by a dominant-negative mutant of Nek2. These studies highlight the importance of protein phosphorylation in the control of centrosome cohesion, and they point to Nek2 and PP1α as critical regulators of centrosome structure.

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