Quantitative analysis of sea urchin egg kinesin-driven microtubule motility

We have analyzed the effects of various substrates and inhibitors on the rates of microtubule (MT) motility induced by sea urchin egg kinesin using real-time computer analysis and video-enhanced light microscopy. In the presence of magnesium, 10 mM concentrations of all the nucleotides tested supported MT translocation, with velocities in MgATP greater than MgGTP greater than MgTTP approximately equal to MgUTP greater than MgCTP greater than MgITP. The velocity of kinesin-driven MT motility is fairly uniform over approximately 3 pH units, from pH 6 to 9, with almost no motility outside this range. In the presence of ATP, no motility is observed in the absence of divalent cations; addition of Mg2+ but not addition of Ca2+ restores motility. MgATP-dependent MT motility is reversibly inhibited by Mg-free ATP, EDTA, or tripolyphosphate, suggesting that Mg-free ATP is an inactive substrate analogue. MgATP and MgGTP both obey saturable, Michaelis-Menten kinetics, with apparent Km values of approximately 60 microM and 2 mM, and Vmax values of approximately 0.6 and 0.4 microns/s, respectively. MgATP gamma S and MgADP are classic competitive inhibitors of kinesin-driven motility in MgATP, with Ki values of approximately 15 and 150 microM, respectively. Adenosine 5'-(beta, gamma-methylene)-triphosphate and N-ethylmaleimide only inhibit MT motility weakly, while adenyl-5'-yl imidodiphosphate and vanadate strongly inhibit MT motility, but not in a simple competitive manner. Moreover, in contrast to other inhibitors which cause a unimodal decrease in MT mean velocity, vanadate concentrations greater than approximately 10% that of MgATP cause some MTs to become immotile, resulting in a bimodal distribution of MT velocities.     

Light chains of sea urchin kinesin identified by immunoadsorption

Previous studies with monoclonal antibodies indicate that sea urchin kinesin contains two heavy chains arranged in parallel such that their N-terminal ends fold into globular mechanochemical heads attached to a thin stalk ending in a bipartite tail [Scholey et al., 1989]. In the present, complementary study, we have used the monoclonal antikinesin, SUK4, to probe the quaternary structure of sea urchin (Strongylocentrotus purpuratus) kinesin. Kinesin prepared from sea urchin cytosol sedimented at 9.6 S on sucrose density gradients and consisted of 130-kd heavy chains plus an 84-kd/78 kd doublet (1 mol heavy chain: 1 mol doublet determined by gel densitometry). Low levels of 110-kd and 90-kd polypeptides were sometimes present as well. The 84-kd/78 kd polypeptides are thought to be light chains because they were precipitated from the kinesin preparation at a stoichiometry of one mol doublet per 1 mol heavy chain using SUK4-Sepharose immunoaffinity resins. The 110-kd and 90-kd peptides, by contrast, were removed using this immunoadsorption method. SUK4-Sepharose immunoaffinity chromatography was also used to purify the 130-kd heavy chain and 84-kd/78-kd doublet (1 mol heavy chain: 1 mol doublet) directly from sea urchin egg cytosolic extracts, and from a MAP (microtubule-associated protein) fraction eluted by ATP from microtubules prepared in the presence of AMPPNP but not from microtubules prepared in ATP. The finding that sea urchin kinesin contains equimolar quantities of heavy and light chains, together with the aforementioned data on kinesin morphology, suggests that native sea urchin kinesin is a tetramer assembled from two light chains and two heavy chains.     

Characterization of sea urchin egg endoplasmic reticulum in cortical preparations

The cortical endoplasmic reticulum (ER) of sea urchin eggs was localized on isolated egg cortices by staining with aqueous suspensions of the dicarbocyanine "DiI." Immunofluorescence localization of a calsequestrin-like protein was essentially identical; this is consistent with a role for the ER in calcium regulation. The ER often encircles cortical granules, making it well-suited for initiating fusion and propagating the calcium wave. Thiazole orange and Hoechst dye 33258 at pH 2 stain ribosomes bound to the ER, providing evidence that the cortical ER is rough ER. High chloride concentrations were found to disrupt ER continuity.     

Fluctuation in the microtubule sliding movement driven by kinesin in vitro.

We studied the fluctuation in the translational sliding movement of microtubules driven by kinesin in a motility assay in vitro. By calculating the mean-square displacement deviation from the average as a function of time, we obtained motional diffusion coefficients for microtubules and analyzed the dependence of the coefficients on microtubule length. Our analyses suggest that 1) the motional diffusion coefficient consists of the sum of two terms, one that is proportional to the inverse of the microtubule length (as the longitudinal diffusion coefficient of a filament in Brownian movement is) and another that is independent of the length, and 2) the length-dependent term decreases with increasing kinesin concentration. This latter term almost vanishes within the length range we studied at high kinesin concentrations. From the length-dependence relationship, we evaluated the friction coefficient for sliding microtubules. This value is much larger than the solvent friction and thus consistent with protein friction. The length independence of the motional diffusion coefficient observed at sufficiently high kinesin concentrations indicates the presence of correlation in the sliding movement fluctuation. This places significant constraint on the possible mechanisms of the sliding movement generation by kinesin motors in vitro.     

Polarized microtubule gliding and particle saltations produced by soluble factors from sea urchin eggs and embryos

In this report, we describe an in vitro system for analyzing microtubule-based movements in supernatants of sea urchin egg and embryo homogenates. Using video enhanced DIC microscopy, we have observed bidirectional saltatory particle movements on native taxol-stabilized microtubules assembled in low speed supernatants of Lytechinus egg homogenates, and gliding of these microtubules across a glass surface. A high speed supernatant of soluble proteins, depleted of organelles, microtubules, and their associated proteins supports the gliding of exogenous microtubules and translocation of polystyrene beads along these microtubules. The direction of microtubule gliding has been determined directly by observation of the gliding of flagellar axonemes in which the (+) and (-) ends could be distinguished by biased polar growth of microtubules off the ends. Microtubule gliding is toward the (-) end of the microtubule, is ATP sensitive, and inhibited only by high concentrations of vanadate. These characteristics suggest that the transport complex responsible for microtubule gliding in S2 is kinesin-like. The implications of these molecular interactions for mitosis and other motile events are discussed.     

Cytoskeleton of the unfertilized sea urchin egg

Unfertilized Paracentrotus lividus egg cytoskeleton is prepared by mild, nonionic detergent extraction at 4 degrees C in buffer systems containing either 2-methyl-2,4-pentanediol (hexylene glycol) or glycerol. These extractions allow the isolation of cytomatrices that maintain the egg form and are 70-80 micron in diameter. DNase inhibition assays show that actin is in polymerized form in these cytomatrices. Ultrastructural observations reveal that the cytoskeletons are made up essentially of 2 categories of filaments, 7-8-nm and 2-4-nm in diameter, respectively. After heavy meromyosin labelling, short, radiating actin filaments are seen in the cortical region, while longer actin filaments are found in the internal region of these cytomatrices. The 2-4-nm filaments of still unknown biochemical nature are organized in a meshwork. In contrast to results found with fertilized eggs, bundles of actin filaments and microtubules are absent; 8-13-nm filaments are not detected.     

Analysis of microtubule polymerization inhibitors in sea urchin egg extracts: Evidence for a protease

Inhibitors of microtubule polymerization have been found in extracts of unfertilized sea urchin eggs using neural tubulin polymerization assays without glycerol. The inhibitory activity is partially destroyed by boiling or by reduction and carboxymethylation and is nondialyzable. When chromatographed on DEAE-cellulose, the inhibitory activity is eluted over a broad NaCl gradient and is in association with several peaks. This partially purified inhibitor is not destroyed by incubation with RNase A. When the partially purified inhibitor is incubated with brain microtubule protein under conditions which support microtubule polymerization, both high molecular weight-microtubule associated proteins and tubulin appear to be digested when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteolytic digestion as well as inhibition of microtubule polymerization depend upon similar concentrations of partially purified inhibitor present in the polymerization reaction. It appears as though at least part of the microtubule polymerization inhibitory activity present in unfertilized sea urchin eggs is due to this protease.     

Buffer conditions and non-tubulin factors critically affect the microtubule dynamic instability of sea urchin egg tubulin.

The dynamic instability of individual microtubules (Mts) in cytoplasmic extracts or assembled from highly purified sea urchin egg tubulin was examined using video-enhanced, differential-interference contrast (VE-DIC) light microscopy. Extract Mts (endogenous tubulin = 12.1 microM) displayed only plus-ended growth. The elongation velocity was 7.8 microns/min for an average duration of 1.3 min before switching (catastrophe) to rapid shortening, which occurred at 13.0 microns/min for an average duration of 0.5 min before switching (rescue) back to the elongation phase. These parameters are typical of interphase Mt dynamic instability. Surprisingly, Mts assembled from purified urchin egg tubulin in standard buffers were less dynamic that those reported for purified brain tubulin or Mts in the extract. Buffer parameters were changed in an attempt to mimic the extract Mt results. The pH buffer itself, Hepes or Pipes, drastically altered Mt dynamics but could not achieve high elongation velocity with high catastrophe frequencies. Calcium at 1 microM had negligible effects, while increasing pH from 6.9 to 7.2 stimulated elongation velocity. Finally, Mt dynamics of purified egg tubulin (11.9 microM) were assayed in ultrafiltrates (MW cut-off less than 30 kD) of the cytoplasmic extracts. Mts elongated slowly at 1.2 microns/min for 26 min before a catastrophe and rapid shortening at 11.8 microns/min. Rescue was less frequent than unfiltered extracts, minus-ended growth was observed, and self-assembly occurred at slightly higher tubulin concentrations. Therefore, the egg extracts and cytoplasm must contain non-buffer factors which stimulate elongation velocity by 6.5-fold without self-assembly, increase catastrophe frequency by 20-fold, and block minus-ended growth.     

Membrane potential of the unfertilized sea urchin egg

The membrane potential, specific resistance, and potassium selectivity of the unfertilized Strongylocentrotus purpuratus egg were determined by two independent methods: tracer flux and microelectrode. The potassium influx was 0.50 ± 0.2 pmole/cm²· sec, which was greater than the sodium, chloride, and calcium influxes by factors of 4, 7, and 75, respectively. By means of the constant-field equations, the flux data were used to calculate membrane potential (?70 mV) and specific resistance (420 kΩ · cm²). The effect of the external potassium concentration on the sodium influx was determined and the results closely fit the result expected if the membrane behaved as a potassium electrode. Microelectrode measurements of the potential and resistance were ?75 ± 3 mV and 380 ± kΩ · cm².     

Inhibitory factor of microtubule assembly from sea urchin egg cortex. I. Preparation and characterization

A new inhibitory factor of the microtubule (MT) assembly system was isolated from unfertilized sea urchin egg cortex. This factor not only suppressed spontaneous brain MT assembly, but also induced depolymerization of the reconstituted MTs. The factor did not suppress initial MT growth initiated by ciliary outer fiber fragments but the assembled MTs were soon depolymerized with time. The inhibitory activity was heat-stable but sensitive to trypsin or urea. The mode of the inhibition was distinct from the inhibitory effects of RNA on the MT assembly. The inhibitory factor partially purified on DEAE-Sephadex A-50 completely inhibited tubulin polymerization in a factor: tubulin ratio of 0.013.     

Proteins of the sea urchin egg vitelline layer

The vitelline layers (VL) of unfertilized sea urchin eggs were isolated, and the diversity of their polypeptide constitutents estimated by two-dimensional polyacrylamide gel electrophoresis. At least 25 components are reproducibly observed. While VL polypeptides are almost certainly synthesized in the growing oocyte, they are not among the more prevalent newly synthesized proteins detected in oocytes that were isolated and labeled in vitro for 4 hr. A set of monoclonal antibodies was raised against VL components and partially characterized. The 31 monoclonals analyzed fell into 11 classes with respect to their avidity for VL proteins solubilized under mild and under strongly denaturing conditions, and to their reactions with surface components of the VLs of living eggs. Fluorescence microscopy showed diverse patterns of surface reactivity when different monoclonal antibodies were compared. Two of the monoclonal antibodies reacted with specific sets of three proteins each on VL protein blots. It is concluded that the VL is a complex structure containing a large number of different polypeptide components, the genes for several of which should now be experimentally accessible.     

Substructure of sea urchin egg cytoplasmic dynein

The substructure of the cytoplasmic dynein molecule was studied using the quick-freeze, deep-etch technique. Cytoplasmic dynein purified as a 12 S form from the eggs of the sea urchin Hemicentrotus pulcherrimus was composed of a single high molecular weight polypeptide. Rotary shadowing images of cytoplasmic dynein either sprayed on to a mica surface or quick-frozen on mica flakes demonstrated a single-headed molecule, in contrast to the two-headed molecule of sea urchin sperm flagellar 21 S dynein. More detailed substructure was visualized by rotary shadowing after quick-freeze deep-etching. Cytoplasmic dynein consisted of a head and a stem. The head was pear-shaped (16 nm X 11 nm) and a little smaller than the pear-shaped head of 21 S dynein (18 nm X 14 nm). The form of the stem was irregular, and its apparent length varied from 0 to 32 nm. Binding of cytoplasmic dynein to brain microtubule in the solution was observed by negative staining, and that in the precipitate was examined by the quick-freeze, deep-etch method as well. Both methods revealed the presence of two kinds of microtubules, one a fully decorated microtubule and the other a non-decorated microtubule. Cytoplasmic dynein bound to microtubule also appeared as a globular particle. Neither the periodic binding nor the crossbridges that were observed with 21 S dynein were formed by cytoplasmic dynein, although cytoplasmic dynein appeared to bind to microtubules co-operatively.     

Coagulation of sea urchin egg cytoplasm by high salt concentrations

Unfertilized sea urchin eggs exposed to hyperosmotic salt solutions in excess of 1.75 M undergo a form of intracellular coagulation known as black cytolysis, similar to that seen in eggs injured by freezing. The process can be simulated by the microinjection of hypertonic salt into the cell suspended in isotonic solution in the absence of volume reduction. Black cytolysis during hyperosmotic stress can be attributed to the entry of concentrated extracellular solution through a membrane made permeable by excessive osmotic stress.