Ionic and nucleotide requirements for microtubule polymerization in vitro

Cell volume homeostasis is vital for the maintenance of optimal protein density and cellular function. Numerous mammalian cell types are routinely exposed to acute hypertonic challenge and shrink. Molecular crowding modifies biochemical reaction rates and decreases macromolecule diffusion. Cell volume is restored rapidly by ion influx but at the expense of elevated intracellular sodium and chloride levels that persist long after challenge. Although recent studies have highlighted the role of molecular crowding on the effects of hypertonicity, the effects of ionic imbalance on cellular trafficking dynamics in living cells are largely unexplored. By tracking distinct fluorescently labeled endosome/vesicle populations by live-cell imaging, we show that vesicle motility is reduced dramatically in a variety of cell types at the onset of hypertonic challenge. Live-cell imaging of actin and tubulin revealed similar arrested microfilament motility upon challenge. Vesicle motility recovered long after cell volume, a process that required functional regulatory volume increase and was accelerated by a return of extracellular osmolality to isosmotic levels. This delay suggests that, although volume-induced molecular crowding contributes to trafficking defects, it alone cannot explain the observed effects. Using fluorescent indicators and FRET-based probes, we found that intracellular ATP abundance and mitochondrial potential were reduced by hypertonicity and recovered after longer periods of time. Similar to the effects of osmotic challenge, isovolumetric elevation of intracellular chloride concentration by ionophores transiently decreased ATP production by mitochondria and abated microfilament and vesicle motility. These data illustrate how perturbed ionic balance, in addition to molecular crowding, affects membrane trafficking.electrolyte | cell volume | hypertonicity | membrane trafficking | mitochondria

Section: Discussionsupporting

confidence: 66%

Section: Significancementioning

confidence: 99%

Ionic imbalance, in addition to molecular crowding, abates cytoskeletal dynamics and vesicle motility during hypertonic stress

Nunes

Roth

Meda

et al. 2015

“…4A and B). After an initial lag the formation of microtubules increases linearly for [8][9][10] min. The polymerization-dependent GTPase also shows a brief lag, then a linear increase (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Although investigators have been cautious about proposing a role for GTP in polymerization, the first polymerization of tubulin in vitro from crude extracts of brain contained GTP (5,6). Additional studies in several laboratories (7)(8)(9) with more homogeneous tubulin preparations demonstrated that GTP was required for polymerization and that GTP analogs, guanylylmethylenediphosphate [GMP-P(CH2)P] and guanylylimidodiphosphate [GMP-P(NH)P], were inactive. These results suggested that hydrolysis of the y-phosphate may be necessary for microtubule formation.…”

mentioning

confidence: 99%

Stoichiometry of GTP hydrolysis and tubulin polymerization.

Maccioni¹,

Seeds²

1977

Microtubule formation from lamb brain tubulin'isolated by affinity chromatography and freed of exchangeable nucleotide requires GTP for maximal rate and extent of polymerization. The nucleotide analogs guanylylmethyleniediphosphate and guanylylimidodiphosphate fail to replace GTP; in addition, neither the presence of mierotubule associated proteins nor 5 M glycerol relieves the GTP requirement. The relation of GTP concentration and microtubule formation shows an association constant K = 1 X 104 M-1; furthermore, GDP and guanylylimidodiphosphate are competitive inhibitors of GTP for polymerization.'Using a rapid filter assay for microtubule formation that allows the quantitative analysis of early polymerization kinetics and correcting for GTP hydrolysis uncoupled from tubulin polymerization, a stoichiometry of two molecules of GTP hydrolyzed per mole of tubulin dimer incorporated into microtubules has been found. (7) and sedimentation (12) assays, respectively. Using this kinetic assay it is now possible to determine the stoichiometry of GTP hydrolysis and tubulin polymerization. MATERIALS AND METHODSMaterials.['y-32P]GTP (30 Ci/mmol) was purchased from New England Nuclear (Boston). Nonradioactive GTP (Types II and IV), ATP, and cyclic 3':5'-GMP (cGMP) were purchased from Sigma Chemical (St. Louis); other nucleotides [GMP, GDP, GMP-P(NH)P and GMP-P(CH2)P] were obtained from P-L Biochemicals (Milwaukee). All other reagents were of the most pure analytical grade available.Preparation of Tubulin. Microtubules were isolated by two cycles of polymerization from lamb brain homogenates as described (9). The crude microtubule pellets were stored at -750 without resuspension until further use. Tubulin was isolated from the microtubule pellet by deacetylcolchicinic acid (DAC)-affinity chromatography as described (9, 13). The purified tubulin was desalted by Sephadex G-25 column chromatography and concentrated in an Amicon PM-10 ultrafiltration apparatus. The concentrated tubulin was centrifuged at 14,000 X g to remove aggregates and used immediately. The protein concentration was determined by the procedure of Lowry et al. (14).Nucleotide Removal. Tubulin preparations were routinely freed of unbound nucleotide or nucleotide bound to the exchangeable site by two successive extractions with 0.5 ml of a 1 mM EDTA, charcoal (prewashed in imidazole-glycerol buffer containing 2% serum albumin, followed by a wash in only imidazole-glycerol buffer) solution per ml of tubulin sample.The residual guanine nucleotide retained by these char-

“…Low temperature (40) can both prevent and disrupt the tubulin assembled with Gmp(CH2)pp although disruption proceeds much more slowly than when GTP is used. At physiological concentrations (0.5-1 mM) of Mg, the polymerization of tubulin "in vitro" has an essential requirement for nucleoside triphosphates (1,2) and for high molecular weight basic (HMWB) proteins (3). Recently, Arai and Kaziro (4) have assembled microtubules in the presence of guanylyl imidodiphosphate [Gmpp(NH)p] that are relatively resistant to disruption by millimolar concentrations of calcium, conditions that cause extensive disruption of the microtubules assembled in GTP.…”

mentioning

confidence: 99%

Role of nucleotides in tubulin polymerization: effect of guanylyl 5'-methylenediphosphonate.

Sandoval¹,

MacDonald²,

Jameson³

et al. 1977

Incubation of 48,000 X g rat brain supernatants for 30 min at 37 degrees with 1-2 mM guanylyl 5'-methylenediphosphonate [Gmp(CH2)pp] results in polymerization of 95-98% of the tubulin present. This is considerably more than the 50% polymerization that can be achieved with the natural nucleotide, GTP, under optimal conditions. Gmp(CH2)pp is also much more effective than GTP in inducing polymerization of purified tubulin. Assembly of microtubules with Gmp(CH2)pp occurs at tubulin concentrations one-third of those possible with GTP. Furthermore, with Gmp(CH2)pp, microtubule assembly does not require the high molecular weight basic proteins needed with GTP. Polymerization of tubulin by Gmp(CH2)pp is neither prevented nor reversed by concentrations of calcium (2 mM) that can either prevent microtubule assembly or disrupt already formed microtubules if the nucleotide used is GTP or guanylyl imidodiphosphate. When Ca2+ is added before or after microtubule assembly, electron microscopy of the Gmp(CH2)pp preparations reveals normal microtubules turning into twisted ribbons. Low temperature (4 degrees) can both prevent and disrupt the tubulin assembled Gmp(CH2)pp although disruption proceeds much more slowly when GTP is used.