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G B Witman

Publications and source records attributed to G B Witman.

At least 73 records · Page 4Linked to original sources
PubMed1984

The photoaffinity probe 8-azidoadenosine 5'-triphosphate selectively labels the heavy chain of Chlamydomonas 12 S dynein.

Chlamydomonas 12 S dynein, which makes up part of the outer arm of the flagellar axoneme, consists of three polypeptides of 330,000, 22,000, and 18,000 daltons. We have used 8-azidoadenosine 5'-triphosphate (8-N3ATP), a photoaffinity analog of ATP, to investigate which of the dynein polypeptides contains the site of ATP hydrolysis. 8-N3ATP is a competitive inhibitor of the hydrolysis of ATP by 12 S dynein and is hydrolyzed by 12 S dynein in an ATP- and vanadate-sensitive fashion, indicating that it binds to the 12 S dynein hydrolytic site in the same way as ATP. When dynein was incubated with [gamma-32P]- or [alpha-32P]8-N3ATP in the presence of UV light to activate the azido moiety, the analog was incorporated into 12 S dynein's heavy polypeptide chain, but not its light chains. The incorporation was UV-dependent, was blocked by addition of ATP or vanadate plus ADP to the reaction mixture, and did not occur in heat-denatured dynein. These results strongly suggest that the hydrolytic site of 12 S dynein is contained in its heavy chain.

Adenosine Triphosphatases↗
PubMed1984

Submicromolar levels of calcium control the balance of beating between the two flagella in demembranated models of Chlamydomonas.

When detergent-extracted, demembranated cell models of Chlamydomonas were resuspended in reactivation solutions containing less than 10(-8) M Ca++, many models initially swam in helical paths similar to those of intact cells; others swam in circles against the surface of the slide or coverslip. With increasing time after reactivation, fewer models swam in helices and more swam in circles. This transition from helical to circular swimming was the result of a progressive inactivation of one of the axonemes; in the extreme case, one axoneme was completely inactive whereas the other beat with a normal waveform. At these low Ca++ concentrations, the inactivated axoneme was the trans-axoneme (the one farthest from the eyespot) in 70-100% of the models. At 10(-7) or 10(-6) M Ca++, cell models also proceeded from helical to circular swimming as a result of inactivation of one of the axonemes; however, under these conditions the cis-axoneme was usually the one that was inactivated. At 10(-8) M Ca++, most cells continued helical swimming, indicating that both axonemes were remaining relatively active. The progressive, Ca++-dependent inactivation of the trans- or cis-axoneme was reversed by switching the cell models to higher or lower Ca++ concentrations, respectively. A similar reversible, selective inactivation of the trans-flagellum occurred in intact cells swimming in medium containing 0.5 mM EGTA and no added Ca++. The results show that there are functional differences between the two axonemes of Chlamydomonas. The differential responses of the axonemes to submicromolar concentrations of Ca++ may form the basis for phototactic turning.

Calcium↗
PubMed1984

Flagellar waveform and rotational orientation in a Chlamydomonas mutant lacking normal striated fibers.

The Chlamydomonas mutant vfl-3 lacks normal striated fibers and microtubular rootlets. Although the flagella beat vigorously, the cells rarely display effective forward swimming. High speed cinephotomicrography reveals that flagellar waveform, frequency, and beat synchrony are similar to those of wild-type cells, indicating that neither striated fibers nor microtubular rootlets are required for initiation or synchronization of flagellar motion. However, in contrast to wild type, the effective strokes of the flagella of vfl-3 may occur in virtually any direction. Although the direction of beat varies between cells, it was not observed to vary for a given flagellum during periods of filming lasting up to several thousand beat cycles, indicating that the flagella are not free to rotate in the mature cell. Structural polarity markers in the proximal portion of each flagellum show that the flagella of the mutant have an altered rotational orientation consistent with their altered direction of beat. This implies that the variable direction of beat is not due to a defect in the intrinsic polarity of the axoneme, and that in wild-type cells the striated fibers and/or associated structures are important in establishing or maintaining the correct rotational orientation of the basal bodies to ensure that the inherent functional polarity of the flagellum results in effective cellular movement. As in wild type, the flagella of vfl-3 coordinately switch to a symmetrical, flagellar-type waveform during the shock response (induced by a sudden increase in illumination), indicating that the striated fibers are not directly involved in this process.

Cell Movement↗
PubMed1983

Outer doublet heterogeneity reveals structural polarity related to beat direction in Chlamydomonas flagella.

Analysis of serial cross-sections of the Chlamydomonas flagellum reveals several structural asymmetries in the axoneme. One doublet lacks the outer dynein arm, has a beak-like projection in its B-tubule, and bears a two-part bridge that extends from the A-tubule of this doublet to the B-tubule of the adjacent doublet. The two doublets directly opposite the doublet lacking the arm have beak-like projections in their B-tubules. These asymmetries always occur in the same doublets from section to section, indicating that certain doublets have consistent morphological specializations. These unique doublets give the axoneme an inherent structural polarity. All three specializations are present in the proximal portion of the axoneme; based on their frequency in random cross-sections of isolated axonemes, the two-part bridge and the beak-like projections are present in the proximal one quarter and one half of the axoneme, respectively, and the outer arm is absent from the one doublet greater than 90% of the axoneme's length. The outer arm-less doublet of each flagellum faces the other flagellum, indicating that each axoneme has the same rotational orientation relative to the direction of its effective stroke. This strongly suggests that the direction of the effective stroke is controlled by a structural component within the axoneme. The striated fibers are associated with specific triplets in a manner suggesting that they play a role in setting up or maintaining the 180 degrees rotational symmetry of the two flagella.

Chlamydomonas↗
PubMed1982

Purification and polypeptide composition of dynein ATPases from Chlamydomonas flagella.

Extraction of isolated, demembranated flagellar axonemes of Chlamydomonas reinhardii with 0.6 M KCl solubilized 77-92% of the total axonemal Mg++ or Ca++-ATPase activity, which sedimented as 18S and 12S peaks in sucrose density gradients. The ATPases of these two peaks were further purified by hydroxyapatite (HAP) column chromatography. The ATPase activity of the 18S peak eluted from the HAP column as a single peak coinciding with the protein peak. The HAP purified 18S ATPase had a specific activity of approximately 2.0 +/- 0.5 mumoles Pi hydrolyzed min/mg and was associated with four high molecular weight (HMW) polypeptides of approximately 310,000-340,000 daltons, two intermediate molecular weight (IMW) polypeptides of 78,000 and 69,000 daltons, and eight low molecular weight (LMW) polypeptides of 7,800-19,600 daltons. When the 12S sucrose gradient peak together with a trailing shoulder were chromatographed on HAP, the ATPase activity was eluted in two peaks designated 12S and 10.5S on the basis of the sedimentation properties of their associated polypeptides. The 12S peak contained a single dynein ATPase having a specific activity of approximately 0.6 +/- 0.3 mumoles Pi hydrolyzed min/mg and associated with approximately 330,000-, 21,700-, and 18,100-dalton polypeptides. The 10.5S peak contained several high, intermediate, and low molecular weight polypeptides; of these, one HMW polypeptide and one 28,700-dalton polypeptide correlated well with the ATPase activity. The purified ATPases had no polypeptides in common; each therefore represents a discrete dynein. Based on protein recovered in the purified fractions, 18S dynein represents approximately 9.2% of the total axonemal protein; 12S dynein represents approximately 4.7% of the axonemal protein.

Adenosine Triphosphatases↗
PubMed1982

Synthesis, transport, and utilization of specific flagellar proteins during flagellar regeneration in Chlamydomonas.

We labeled gametes of Chlamydomonas with 10-min pulses of 35SO4(-2) before and at various times after deflagellation, and isolated whole cells and flagella immediately after the pulse. The labeled proteins were separated by one- or two-dimensional gel electrophoresis, and the amount of isotope incorporated into specific proteins was determined. Individual proteins were identified with particular structures by correlating missing axonemal polypeptides with ultrastructural defects in paralyzed mutants, or by polypeptide analysis of flagellar fractions. Synthesis of most flagellar proteins appeared to be coordinately induced after flagellar amputation. The rate of synthesis for most quantified proteins increased at least 4- to 10-fold after deflagellation. The kinetics of synthesis of proteins contained together within a structure (e.g., the radial spoke proteins [RSP] ) were frequently similar; however, the kinetics of synthesis of proteins contained in different structures (e.g., RSP vs. alpha- and beta-tubulins) were different. Most newly synthesized flagellar proteins were rapidly transported into the flagellum with kinetics reflecting the rate of growth of the organelle; exceptions included a central tubule complex protein (CT1) and an actinlike component, both of which appeared to be supplied almost entirely from pre-existing, unlabeled pools. Isotope dilution experiments showed that, for most quantified axonemal proteins, a minimum of 35-40% of the polypeptide chains used in assembling a new axoneme was synthesized during regeneration; these proteins appeared to have predeflagellation pools of approximately the same size relative to their stoichiometries in the axoneme. In contrast, CT1 and the actinlike protein had comparatively large pools.

Chlamydomonas↗
PubMed1982

Dynein arm conformation and mechanochemical transduction in the eukaryotic flagellum.

When partially disrupted axonemes of Chlamydomonas were negatively stained in the presence of ATP, the dynein arms appeared club-shaped and were tilted proximally. In the absence of ATP, or in the presence of the non-hydrolysable ATP analogue, adenylyl imidodiphosphate, the arms on most doublets appeared to be made up of a thin diagonal component plus a larger component which resembled the arms seen in the presence of ATP but was more upright. Under these same conditions, the arms on some doublets appeared as distally directed ellipses. The two different images observed in the absence of ATP were interconvertible by tilting the doublets in a goniometer stage, indicating that they represented arms in the same conformational state viewed from two different angles. A three-dimensional model has been constructed which accounts for the appearance of the arms at the different angles. Tilting did not convert either of these images into those seen in the presence of ATP. Examination of mutants lacking the inner and outer arms indicated that all of the images were derived from the outer arm. In the absence of ATP, arms were frequently observed which were bound to a B tubule but had lost their attachment to the A tubule; such arms were not observed in the presence of ATP. These observations provide direct evidence for an ATP-dependent conformational change in the outer arm, and suggest a sequence of steps for the mechanochemical cycle of this arm.

Adenosine Triphosphatases↗
PubMed1981

Functionally significant central-pair rotation in a primitive eukaryotic flagellum.

There is now considerable evidence that the basis for ciliary and flagellar movement is an active sliding between peripheral doublet microtubules which, when resisted by structures within the axoneme, leads to axonemal bend formation. In contrast, relatively little is known about the control mechanisms which coordinate the interdoublet sliding and axonemal binding to produce the effective motion observed in various cilia and flagella. One component of the axoneme which may be involved in this control is the central pair of microtubules. To learn more about the action of the central pair, we have studied the tiny uniflagellate marine alga, Micromonas pusilla. The central tubules of the M. pusilla flagellum extend for several micrometres beyond the termination of the peripheral doublets, thus permitting direct observation of the central pair during flagellar movement. Our findings, reported here, indicate that in living M. pusilla the central pair of microtubules undergoes continuous rotation in one direction. This rotation provides the motive force for the cell.

Cell Membrane↗
PubMed1980

Calcium control of waveform in isolated flagellar axonemes of Chlamydomonas.

The effect of Ca(++) on the waveform of reactivated, isolated axonemes of chlamydomonas flagella was investigated. Flagella were detached and isolated by the dibucaine procedure and demembranated by treatment with the detergent Nonidet; the resulting axomenes lack the flagellar membrane and basal bodies. In Ca(++)-buffered reactivation solutions containing 10(-6) M or less free Ca(++), the axonemes beat with a highly asymmetrical, predominantly planar waveform that closely resembled that of in situ flagella of forward swimming cells. In solutions containing 10(-4) M Ca(++), the axonemes beat with a symmetrical waveform that was very similar to that of in situ flagella during backward swimming. In 10(-5) M Ca(++), the axonemes were predominantly quiescent, a state that appears to be closely associated with changes in axomenal waveform or direction of beat in many organisms. Experiments in which the concentrations of free Ca(++), not CaATP(--) complex were independently varied suggested that free Ca(++), not CaATP(--), was responsible for the observed changes. Analysis of the flagellar ATPases associated with the isolated axonemes and the nonidet- soluble membrane-matrix fraction obtained during preparation of the axonemes showed that the axonemes lacked the 3.0S Ca(++)-activated ATPase, almost all of which was recovered in the membrane-matrix fraction. These results indicate that free Ca(++) binds directly to an axonemal component to alter flagellar waveform, and that neither the 3.0S CaATPase nor the basal bodies are directly involved in this change.

Adenosine Triphosphatases↗
PubMed1980

Purification of calmodulin from Chlamydomonas: calmodulin occurs in cell bodies and flagella.

Calmodulin has been purified from cell bodies of the green alga Chlamydomonas by Ca++-dependent affinity chromatography on fluphenazine-Sepharose 4B. Calmodulin from this primitive organism closely resembles that from bovine brain in a number of properties, including (a) binding to fluphenazine in a Ca++-dependent, reversible manner, (b) functioning as a heat-stable, Ca++-dependent activator of cyclic nucleotide phosphodiesterase, and (c) electrophoretic mobility in SDS-polyacrylamide gels in both the presence and absence of Ca++, which causes a shift in the relative mobility of calmodulin. Calmodulin has also been identified by the criteria of phosphodiesterase activation and electrophoretic mobility in both the detergent soluble "membrane plus matrix" and the axoneme fractions of Chlamydomonas flagella. Calmodulin is not associated with the partially purified 12S or 18S dynein ATPases of Chlamydomonas. The presence of calmodulin in the flagellum suggests that it is involved in one or more of the Ca++-dependent activities of this organelle.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1978

Chlamydomonas flagellar mutants lacking radial spokes and central tubules. Structure, composition, and function of specific axonemal components.

The fine structure, protein composition, and roles in flagellar movement of specific axonemal components were studied in wild-type Chlamydomonas and paralyzed mutants pf-14, pf-15A, and pf-19. Electron microscope examination of the isolated axoneme of pf-14 showed that it lacks the radial spokes but is otherwise structurally normal. Comparison of isolated axonemes of wild type and pf-14 by sodium dodecyl sulfate-acrylamide gel electrophoresis indicated that the mutant is missing a protein of 118,000 mol wt; this protein is apparently a major component of the spokes. Pf-15A and pf-19 lack the central tubules and sheath; axonemes of these mutants are missing three high molecular weight proteins which are probably components of the central tubule-central sheath complex. Under conditions where wild-type axonemes reactivated, axonemes of the three mutants remained intact but did not form bends. However, mutant and wild-type axonemes underwent identical adenosine triphosphate-induced disintegration after treatment with trypsin; the dynein arms of the mutants are therefore capable of generating interdoublet shearing forces. These findings indicated that both the radial spokes and the central tubule-central sheath complex are essential for conversion of interdoublet sliding into axonemal bending. Moreover, because axonemes of pf-14 remained intact under reactivating conditions, the nexin links alone are sufficient to limit the amount of interdoublet sliding that occurs. The axial periodicities of the central sheath, dynein arms, radial spokes, and nexin links of Chlamydomonas were determined by electron microscopy using the lattice-spacing of crystalline catalase as an internal standard. Some new ultrastructural details of the components are described.

Adenosine Triphosphate↗
PubMed1978

Effects of adenylyl imidodiphosphate, a nonhydrolyzable adenosine triphosphate analog, on reactivated and rigor wave sea urchin sperm.

A nonhydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP), has been used to study the role of ATP binding in flagellar motility. Sea urchin sperm of Lytechinus pictus were demembranated, reactivated, and locked in "rigor waves" by a modification of the method of Gibbons and Gibbons (11). Rigor wave sperm relaxed within 2 min after addition of 4 micrometer ATP, and reactivated upon addition of 10-12 micrometer ATP. The beat frequency of the reactivated sperm varied with ATP concentration according to Michaelis-Menten kinetics ("Km" = 0.24 mM; "Vmax" = 44 Hz) and was competitively inhibited by AMP-PNP (Ki" approximately to 8.1 mM). Rigor wave sperm were completely relaxed (straightened) within 2 min by AMP-PNP at concentrations of 2-4 mM. The possibilities that relaxation in AMP-PNP was a result of ATP contamination, AMP-PNP hydrolysis, or lowering of the free Mg++ concentration were conclusively ruled out. The results suggest that dynein cross-bridge release is dependent upon ATP binding but not hydrolysis.

Adenosine Triphosphate↗
PubMed1976

Tubulin requires tau for growth onto microtubule initiating sites.

Tubulin purified by phosphocellulose chromatography and free of accessory proteins will not form microtubules in the absence or presence of microtubule initiating sites (flagellar microtubules). The capacity for growth onto pre-existing "seeds" can be restored by the addition of small quantities of partially purified tau protein. Larger quantities restore the capacity for spontaneous assembly. These results suggest that tubulin requires tau for both initiation and growth of microtubules and that tau is incorporated into the microtubule throughout its length.

Animals↗
PubMed1974

Directionality of brain microtubule assembly in vitro.

Chicks were injected intra-cerebrally with [(3)H]leucine, and (3)H-labeled microtubules were assembled in vitro in the brain supernatants. Pieces of these (3)H-labeled microtubules were then incubated with unlabeled brain tubulin subunits under conditions where the subunits assembled onto the labeled pieces. Electron microscopic autoradiography of the negatively-stained microtubules showed all of the radioactivity at one end of the tubules as they increased in length. This clearly demonstrated that the microtubules of brain in this in vitro system were being assembled unidirectionally.

Animals↗
PubMed1972

Chlamydomonas flagella. I. Isolation and electrophoretic analysis of microtubules, matrix, membranes, and mastigonemes.

Methods were developed for the isolation of Chlamydomonas flagella and for their fractionation into membrane, mastigoneme, "matrix," and axoneme components. Each component was studied by electron microscopy and acrylamide gel electrophoresis. Purified membranes retained their tripartite ultrastructure and were shown to contain one high molecular weight protein band on electrophoresis in sodium dodecyl sulfate (SDS)-urea gels. Isolated mastigonemes (hairlike structures which extend laterally from the flagellar membrane in situ) were of uniform size and were constructed of ellipsoidal subunits joined end to end. Electrophoretic analysis of mastigonemes indicated that they contained a single glycoprotein of approximately 170,000 daltons The matrix fraction contained a number of proteins (particularly those of the amorphous material surrounding the microtubules), which became solubilized during membrane removal. Isolated axonemes retained the intact "9 + 2" microtubular structure and could be subfractionated by treatment with heat or detergent. Increasing concentrations of detergent solubilized axonemal microtubules in the following order: one of the two central tubules; the remaining central tubule and the outer wall of the B tubule; the remaining portions of the B tubule; the outer wall of the A tubule; the remainder of the A tubule with the exception of a ribbon of three protofilaments. These three protofilaments appeared to be the "partition" between the lumen of the A and B tubule. Electrophoretic analysis of isolated outer doublets of 9 + 2 flagella of wild-type cells and of "9 + 0" flagella of paralyzed mutants indicated that the outer doublets and central tubules were composed of two microtubule proteins (tubulins 1 and 2) Tubulins 1 and 2 were shown to have apparent molecular weights of 56,000 and 53,000 respectively

Acrylamides↗
PubMed1972

Chlamydomonas flagella. II. The distribution of tubulins 1 and 2 in the outer doublet microtubules.

Quantitative ultrastructural analysis and quantitative gel electrophoresis of preparations of selectively solubilized Chlamydomonas outer doublets indicated that tubulins 1 and 2 were present in both the A tubule and the B tubule, and that only tubulin 1 was present in the three protofilaments which form the wall ("partition") between the lumens of the A and B tubules. The data suggested that the remaining protofilaments of the outer doublet were grouped together in pairs containing the same type of tubulin, pairs containing tubulin 1 alternating with pairs containing tubulin 2. These findings were used to construct models for the arrangement of the two tubulins in the outer doublet. Further analysis by isoelectric focusing resolved tubulins 1 and 2 into at least five bands.

Acrylamides↗