scholarly journals SSNA1 stabilizes dynamic microtubules and detects microtubule damage

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Elizabeth J Lawrence ◽  
Goker Arpag ◽  
Cayetana Arnaiz ◽  
Marija Zanic
Keyword(s):  
Dynamic Instability ◽  
Direct Effects ◽  
Microtubule Dynamic ◽  
Microtubule Severing ◽  
Slowing Down ◽  
Dividing Cells ◽  
Developing Neurons ◽  
Rates Of Growth ◽  
Dynamic Microtubule

Sjögren's Syndrome Nuclear Autoantigen 1 (SSNA1/NA14) is a microtubule-associated protein with important functions in cilia, dividing cells and developing neurons. However, the direct effects of SSNA1 on microtubules are not known. We employed in vitro reconstitution with purified proteins and TIRF microscopy to investigate the activity of human SSNA1 on dynamic microtubule ends and lattices. Our results show that SSNA1 modulates all parameters of microtubule dynamic instability - slowing down the rates of growth, shrinkage and catastrophe, and promoting rescue. We find that SSNA1 forms stretches along growing microtubule ends and binds cooperatively to the microtubule lattice. Furthermore, SSNA1 is enriched on microtubule damage sites, occurring both naturally, as well as induced by the microtubule severing enzyme spastin. Finally, SSNA1 binding protects microtubules against spastin's severing activity. Taken together, our results demonstrate that SSNA1 is both a potent microtubule stabilizing protein and a novel sensor of microtubule damage; activities that likely underlie SSNA1's functions on microtubule structures in cells.

scholarly journals SSNA1 stabilizes dynamic microtubules and detects microtubule damage

2021 ◽  
Author(s):  
EJ Lawrence ◽  
C Arnaiz ◽  
G Arpag ◽  
M Zanic
Keyword(s):  
Dynamic Instability ◽  
Direct Effects ◽  
Cellular Functions ◽  
Microtubule Dynamic ◽  
Slowing Down ◽  
Dividing Cells ◽  
Developing Neurons ◽  
Rates Of Growth ◽  
Dynamic Microtubule

ABSTRACTSjögren’s Syndrome Nuclear Autoantigen 1 (SSNA1/NA14) is a microtubule-associated protein with important functions in cilia, dividing cells and developing neurons. However, the direct effects of SSNA1 on microtubules are not known. We employed in vitro reconstitution with purified proteins and TIRF microscopy to investigate the activity of human SSNA1 on dynamic microtubule ends and lattices. We find that SSNA1 modulates all parameters of microtubule dynamic instability – slowing down the rates of growth, shrinkage and catastrophe, and promoting rescue. SSNA1 accumulation on dynamic microtubule ends correlates with the growth rate slow-down. Furthermore, SSNA1 prevents catastrophe when soluble tubulin is removed or sequestered by Op18/Stathmin. Finally, SSNA1 detects spastin-induced damage and inhibits spastin’s severing activity. Therefore, SSNA1 is both a potent microtubule stabilizing protein and a sensor of microtubule damage; activities that likely underlie SSNA1’s cellular functions.

Analysis of the Mechanism of Microtubule Dynamic Instability Using a UV Microbeam to Sever Elongating Microtubules

1988 ◽  
Vol 46 ◽  
pp. 122-123
Author(s):  
R.A Walker ◽  
S. Inoue ◽  
E.D. Salmon
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis ◽  
Abrupt Transition ◽  
Microtubule Associated Proteins ◽  
Asymmetrical Structure ◽  
Associated Proteins

Microtubules polymerized in vitro from tubulin purified free of microtubule-associated proteins exhibit dynamic instability (1,2,3). Free microtubule ends exist in persistent phases of elongation or rapid shortening with infrequent, but, abrupt transitions between these phases. The abrupt transition from elongation to rapid shortening is termed catastrophe and the abrupt transition from rapid shortening to elongation is termed rescue. A microtubule is an asymmetrical structure. The plus end grows faster than the minus end. The frequency of catastrophe of the plus end is somewhat greater than the minus end, while the frequency of rescue of the plus end in much lower than for the minus end (4).The mechanism of catastrophe is controversial, but for both the plus and minus microtubule ends, catastrophe is thought to be dependent on GTP hydrolysis. Microtubule elongation occurs by the association of tubulin-GTP subunits to the growing end. Sometime after incorporation into an elongating microtubule end, the GTP is hydrolyzed to GDP, yielding a core of tubulin-GDP capped by tubulin-GTP (“GTP-cap”).

Kinetic stabilization of microtubule dynamic instability in vitro by vinblastine

Biochemistry ◽  
1993 ◽  
Vol 32 (5) ◽  
pp. 1285-1293 ◽  
Author(s):  
Robert J. Toso ◽  
Mary Ann Jordan ◽  
Kevin W. Farrell ◽  
Brian Matsumoto ◽  
Leslie Wilson
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic

scholarly journals Aurora B suppresses microtubule dynamics and limits central spindle size by locally activating KIF4A

2013 ◽  
Vol 202 (4) ◽  
pp. 605-621 ◽  
Author(s):  
Ricardo Nunes Bastos ◽  
Sapan R. Gandhi ◽  
Ryan D. Baron ◽  
Ulrike Gruneberg ◽  
Erich A. Nigg ◽  
...  
Keyword(s):  
Dynamic Instability ◽  
Phosphorylation Site ◽  
Size Control ◽  
Aurora B ◽  
Microtubule Dynamic ◽  
Central Spindle ◽  
Specific Subset ◽  
Mutant Forms

Anaphase central spindle formation is controlled by the microtubule-stabilizing factor PRC1 and the kinesin KIF4A. We show that an MKlp2-dependent pool of Aurora B at the central spindle, rather than global Aurora B activity, regulates KIF4A accumulation at the central spindle. KIF4A phosphorylation by Aurora B stimulates the maximal microtubule-dependent ATPase activity of KIF4A and promotes its interaction with PRC1. In the presence of phosphorylated KIF4A, microtubules grew more slowly and showed long pauses in growth, resulting in the generation of shorter PRC1-stabilized microtubule overlaps in vitro. Cells expressing only mutant forms of KIF4A lacking the Aurora B phosphorylation site overextended the anaphase central spindle, demonstrating that this regulation is crucial for microtubule length control in vivo. Aurora B therefore ensures that suppression of microtubule dynamic instability by KIF4A is restricted to a specific subset of microtubules and thereby contributes to central spindle size control in anaphase.

scholarly journals Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.

1997 ◽  
Vol 8 (6) ◽  
pp. 973-985 ◽  
Author(s):  
R J Vasquez ◽  
B Howell ◽  
A M Yvon ◽  
P Wadsworth ◽  
L Cassimeris
Keyword(s):  
Dynamic Instability ◽  
Light Level ◽  
Pause Duration ◽  
Microtubule Assembly ◽  
Differential Interference Contrast Microscopy ◽  
Microtubule Dynamic ◽  
State Behavior ◽  
Dose Dependent Decrease

Previous studies demonstrated that nanomolar concentrations of nocodazole can block cells in mitosis without net microtubule disassembly and resulted in the hypothesis that this block was due to a nocodazole-induced stabilization of microtubules. We tested this hypothesis by examining the effects of nanomolar concentrations of nocodazole on microtubule dynamic instability in interphase cells and in vitro with purified brain tubulin. Newt lung epithelial cell microtubules were visualized by video-enhanced differential interference contrast microscopy and cells were perfused with solutions of nocodazole ranging in concentration from 4 to 400 nM. Microtubules showed a loss of the two-state behavior typical of dynamic instability as evidenced by the addition of a third state where they exhibited little net change in length (a paused state). Nocodazole perfusion also resulted in slower elongation and shortening velocities, increased catastrophe, and an overall decrease in microtubule turnover. Experiments performed on BSC-1 cells that were microinjected with rhodamine-labeled tubulin, incubated in nocodazole for 1 h, and visualized by using low-light-level fluorescence microscopy showed similar results except that nocodazole-treated BSC-1 cells showed a decrease in catastrophe. To gain insight into possible mechanisms responsible for changes in dynamic instability, we examined the effects of 4 nM to 12 microM nocodazole on the assembly of purified tubulin from axoneme seeds. At both microtubule plus and minus ends, perfusion with nocodazole resulted in a dose-dependent decrease in elongation and shortening velocities, increase in pause duration and catastrophe frequency, and decrease in rescue frequency. These effects, which result in an overall decrease in microtubule turnover after nocodazole treatment, suggest that the mitotic block observed is due to a reduction in microtubule dynamic turnover. In addition, the in vitro results are similar to the effects of increasing concentrations of GDP-tubulin (TuD) subunits on microtubule assembly. Given that nocodazole increases tubulin GTPase activity, we propose that nocodazole acts by generating TuD subunits that then alter dynamic instability.

scholarly journals Regulation of microtubule dynamic instability by the carboxy-terminal tail of β-tubulin

2018 ◽  
Vol 1 (2) ◽  
pp. e201800054 ◽  
Author(s):  
Colby P Fees ◽  
Jeffrey K Moore
Keyword(s):  
Dynamic Instability ◽  
Intrinsic Property ◽  
In Vivo Studies ◽  
Microtubule Dynamic ◽  
Microtubule Stability ◽  
Microtubule Networks ◽  
Carboxy Terminal ◽  
Β Tubulin

Dynamic instability is an intrinsic property of microtubules; however, we do not understand what domains of αβ-tubulins regulate this activity or how these regulate microtubule networks in cells. Here, we define a role for the negatively charged carboxy-terminal tail (CTT) domain of β-tubulin in regulating dynamic instability. By combining in vitro studies with purified mammalian tubulin and in vivo studies with tubulin mutants in budding yeast, we demonstrate that β-tubulin CTT inhibits microtubule stability and regulates the structure and stability of microtubule plus ends. Tubulin that lacks β-tubulin CTT polymerizes faster and depolymerizes slower in vitro and forms microtubules that are more prone to catastrophe. The ends of these microtubules exhibit a more blunted morphology and rapidly switch to disassembly after tubulin depletion. In addition, we show that β-tubulin CTT is required for magnesium cations to promote depolymerization. We propose that β-tubulin CTT regulates the assembly of stable microtubule ends and provides a tunable mechanism to coordinate dynamic instability with ionic strength in the cell.

scholarly journals Asymmetric behavior of severed microtubule ends after ultraviolet-microbeam irradiation of individual microtubules in vitro.

1989 ◽  
Vol 108 (3) ◽  
pp. 931-937 ◽  
Author(s):  
R A Walker ◽  
S Inoué ◽  
E D Salmon
Keyword(s):  
Sea Urchin ◽  
Molecular Basis ◽  
Dynamic Instability ◽  
Second Step ◽  
Microtubule Dynamic ◽  
Asymmetric Behavior ◽  
Ultraviolet Microbeam ◽  
Cap Model ◽  
Flagellar Axoneme

The molecular basis of microtubule dynamic instability is controversial, but is thought to be related to a "GTP cap." A key prediction of the GTP cap model is that the proposed labile GDP-tubulin core will rapidly dissociate if the GTP-tubulin cap is lost. We have tested this prediction by using a UV microbeam to cut the ends from elongating microtubules. Phosphocellulose-purified tubulin was assembled onto the plus and minus ends of sea urchin flagellar axoneme fragments at 21-22 degrees C. The assembly dynamics of individual microtubules were recorded in real time using video microscopy. When the tip of an elongating plus end microtubule was cut off, the severed plus end microtubule always rapidly shortened back to the axoneme at the normal plus end rate. However, when the distal tip of an elongating minus end microtubule was cut off, no rapid shortening occurred. Instead, the severed minus end resumed elongation at the normal minus end rate. Our results show that some form of "stabilizing cap," possibly a GTP cap, governs the transition (catastrophe) from elongation to rapid shortening at the plus end. At the minus end, a simple GTP cap is not sufficient to explain the observed behavior unless UV induces immediate recapping of minus, but not plus, ends. Another possibility is that a second step, perhaps a structural transformation, is required in addition to GTP cap loss for rapid shortening to occur. This transformation would be favored at plus, but not minus ends, to account for the asymmetric behavior of the ends.

Microtubule-associated protein autophosphorylation alters in vitro microtubule dynamic instability

FEBS Letters ◽  
1992 ◽  
Vol 296 (1) ◽  
pp. 21-24 ◽  
Author(s):  
Nadia Raffaelli ◽  
Paul S. Yamauchi ◽  
Daniel L. Purich
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic

Time-Resolved Cryo-Electron Microscopy of Oscillating Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 502-503
Author(s):  
Eva-Maria Mandelkow ◽  
Ron Milligan
Keyword(s):  
Electron Microscopy ◽  
Dynamic Instability ◽  
Entire Solution ◽  
Reaction Scheme ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
A Chain ◽  
Ray Scattering

Microtubules form part of the cytoskeleton of eukaryotic cells. They are hollow libers of about 25 nm diameter made up of 13 protofilaments, each of which consists of a chain of heterodimers of α-and β-tubulin. Microtubules can be assembled in vitro at 37°C in the presence of GTP which is hydrolyzed during the reaction, and they are disassembled at 4°C. In contrast to most other polymers microtubules show the behavior of “dynamic instability”, i.e. they can switch between phases of growth and phases of shrinkage, even at an overall steady state [1]. In certain conditions an entire solution can be synchronized, leading to autonomous oscillations in the degree of assembly which can be observed by X-ray scattering (Fig. 1), light scattering, or electron microscopy [2-5]. In addition such solutions are capable of generating spontaneous spatial patterns [6].In an earlier study we have analyzed the structure of microtubules and their cold-induced disassembly by cryo-EM [7]. One result was that disassembly takes place by loss of protofilament fragments (tubulin oligomers) which fray apart at the microtubule ends. We also looked at microtubule oscillations by time-resolved X-ray scattering and proposed a reaction scheme [4] which involves a cyclic interconversion of tubulin, microtubules, and oligomers (Fig. 2). The present study was undertaken to answer two questions: (a) What is the nature of the oscillations as seen by time-resolved cryo-EM? (b) Do microtubules disassemble by fraying protofilament fragments during oscillations at 37°C?

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