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C J Brokaw

Publications and source records attributed to C J Brokaw.

At least 19 recordsLinked to original sources
PubMed1997

Transient disruptions of axonemal structure and microtubule sliding during bend propagation by Ciona sperm flagella.

Demembranated sperm flagella of Ciona were reactivated at increased salt concentrations (0.45 to 0.5 M K acetate). In addition to a decrease in amplitude of propagated bends, some flagella switch between "stable" and "transient" bending cycles. In the transient bending cycles, there is increased intermicrotubule sliding, in the direction that forms a new principal bend at the base of the flagellum, during the first half of a bending cycle. The magnitude of this increased sliding may be as much as 1 radian, or 0.06 micron between adjacent doublet microtubules. Most transient bending patterns also show a characteristic disruption of axonemal structure, involving separation between strands of microtubule doublets over a distance of up to 5 microns, occurring within a principal bend, typically about 16 microns from the base of the flagellum. The disruptions usually disappear after the principal bend propagates beyond the region of the disruption. Formation of these disruptions requires additional sliding, in the direction that would form a principal bend at the base of the flagellum, of up to about 0.3 micron. Formation of these disruptions may be explained by weakening of structural interactions by increased salt concentration and transverse forces, proportional to curvature and transmitted force, that will tend to separate doublets in a bend. These observations indicate that an actively beating flagellum possesses active sliding capability that is activated but not expressed during normal bend initiation and propagation. The initiation and propagation of flagellar bends may not be explicable solely in terms of local activation and inactivation of dynein-driven sliding.

Animals↗
PubMed1997

Mechanical components of motor enzyme function.

Motor enzymes use energy from ATP dephosphorylation to generate movement by a mechanical cycle, moving and pushing in one direction while attached to their cytoskeletal substrate, and recovering by moving relative to their substrate to a new attachment site. Mainstream models assert that movement while attached to the substrate results from preexisting strain in the attached motor. The additional underlying ideas can be described in terms of three components for strain amplification: a rotating lever arm, multiple attached states, and elastic compliance. These components determine how energy is recovered during the mechanical cycle and stored in a strained motor. They may coexist in a real motor; the challenge is to determine the contributions of each component. Because these components can generate similar relationships between strain energy and strain, standard measurements of motor function do not discriminate easily between these components. However, important information could be is provided by observations that suggest weak coupling between chemical and mechanical cycles, observations of negative force and movement events in single motor experiments, and the discovery that two motors that move in opposite directions have very similar structures. In models incorporating changes in conformation between attached states, these observations are only explained easily if the conformational changes are tightly coupled to changes in the strength of motor-substrate binding.

Actins↗
PubMed1996

Microtubule sliding, bend initiation, and bend propagation parameters of Ciona sperm flagella altered by viscous load.

The effect of altered viscous resistance on flagellar bending has been reexamined, utilizing ATP-reactivated sperm flagella from Ciona and newer methods that resolve metachronous and synchronous components of microtubule sliding and allow the examination of bend initiation as well as bend propagation. Large changes in amplitude and wavelength of bend propagation occur with little change in bend initiation parameters, other than frequency, indicating that bend initiation and bend propagation are regulated by quite different mechanisms. At increased viscosity, reduced amplitude of propagating bends, measured as metachronous shear amplitude, is associated with both reduced amplitude during bend initiation and amplitude adjustment after bends begin to propagate. This combination of effects was seen previously when reduced amplitudes were induced by increased salt concentration, and it was suggested to be caused by an imbalance between active moments and viscous resistances. However, in contrast to the results at increased salt concentrations, which involved significant reduction in bend curvature and little reduction in wavelength, increased viscosity causes very little change in curvature and causes a major reduction in wavelength. This difference can be explained by a model of flagellar bending in which inner arm dyneins have primary responsibility for maintaining bend curvature and outer arm dyneins have primary responsibility for performing work against viscous resistances. Both sets of dyneins would be inhibited by increased salt concentration, but increased viscous resistance would be irrelevant to the operation of inner arm dyneins.

Adenosine Triphosphate↗
PubMed1995

Multiple protein kinase activities required for activation of sperm flagellar motility.

A specific peptide inhibitor of the cyclic AMP (cAMP)-dependent protein kinase (PKI-peptide) is a very effective inhibitor of the cAMP-dependent activation of motility of Ciona spermatozoa, when PKI-peptide is present at the beginning of incubation of demembranated spermatozoa with cAMP and ATP. Under conditions where approximately 120 sec is required for full activation of motility, the window of sensitivity to the PKI-peptide lasts for only 25-30 sec. Examination of sperm pellet proteins labeled with 32P ATP during activation reveals a major 25 kDa phosphoprotein and 2 minor phosphoproteins whose phosphorylation is highly sensitive to to inhibition by the PKI-peptide and essentially complete during this early phase. These sperm proteins appear to be immediate substrates for cAMP-dependent protein kinase, and phosphorylation of one or more of these appears to be requires, but not sufficient, for activation of motility. The phosphorylation of other proteins is reduced or eliminated when PKI-peptide is present at the beginning of incubation, but is unaffected by later addition of PKI-peptide. Some of these substrates appear to be likely candidates for axonemal proteins that must be phosphorylated during the later stages of incubation in order to complete the activation process. This selection is based upon a high degree of inhibition by inclusion of PKI-peptide or other inhibitors at the start of the incubation process, on near-completion of their phosphorylation by the end of the 2 min incubation period required for the activation of motility, and evidence that these proteins are phosphorylated during in vivo activation of motility. Although these observations suggest the presence of a second kinase activity that is upregulated by the initial activation of the cAMP-dependent protein kinase, assays using exogenous substrates have not yet been able to identify such a kinase activity.

Animals↗
PubMed1995

Weakly-coupled models for motor enzyme function.

In strongly-coupled models for motor enzyme function, such as the original Huxley (1957) model for muscle, ATP binding and subsequent hydrolysis are required for the detachment and reattachment of every force-producing cross-bridge. In weakly-coupled models, cross-bridges can be 'mechanically detached' without ATP binding when they have been pushed far beyond their free energy minimum and have accumulated so much strain that the attached state is less stable than the detached state. Weakly-coupled models assume that these mechanically detached cross-bridges can rejoin the pool of detached molecules that can reattach as force-producing cross-bridges, without going through an ATP hydrolysis cycle. This paper bases this assumption on a thermodynamically rigorous model for interaction between a motor enzyme molecule and binding sites on a cytoskeletal protein filament, equivalent to other examples of ligand binding interactions. It attempts to identify more clearly the features that must be added to the idea of ligand binding equilibrium to simulate a weakly-coupled motor enzyme model. Models that assume a vectorial conformational change and a longitudinal series elastic element appear to be incompatible with the assumptions of weakly-coupled cross-bridge models. A stochastic computational method has been used to examine the properties of these models. The computations have examined the behaviour of a model containing a four-state ATPase cycle, but the model is computationally a nine-state model because a force-generating attached state is allowed to equilibrate with different detached states at negative and at positive distortions, and because three adjacent sites are considered as possible attachment sites for each of the two attached states of the ATPase cycle.

Adenosine Triphosphatases↗
PubMed1994

Microtubule sliding in reduced-amplitude bending waves of Ciona sperm flagella: bending waves attenuated by lithium.

The distinct damped, or attenuated, bending pattern observed when demembranated sperm flagella of the tunicate, Ciona, are reactivated in the presence of 2 mM Li+ has been analysed in detail. In these patterns, bends are initiated at the base of the flagellum, but die out after they start to propagate along the flagellum, so that little or no bending is seen in the distal half of the flagellum. A quantitative descriptive analysis shows that the distinctive feature of this attenuation of bending wave amplitude is an asymmetric interbend decay, or slippage, occurring, on average, only at the transitions between a reverse bend and the preceding principal bend. This attenuation is combined with a significant amount of synchronous sliding in the distal half of the flagellum and a decrease in propagation velocity of transitions between bends in the mid-region of the flagellum. Computer simulations demonstrate that the synchronous sliding in the distal half of these flagella can be an entirely passive consequence of the mechanical interaction between active sliding and bending in the basal third of the flagellum and viscous resistances to movement of the distal region of the flagellum through the fluid environment. The current computer models do not contain a mechanism for asymmetric interbend decay that can reproduce these attenuated bending patterns.

Animals↗
PubMed1994

Control of flagellar bending: a new agenda based on dynein diversity.

Observations that were interpreted to provide evidence for equivalent functions of all axonemal dyneins should be reinterpreted, and models based on this assumption should be abandoned. In the future, attempts to understand the mechanisms for flagellar bending, oscillation, and bend propagation should start from the assumption that each type of axonemal dynein may have a specific function. At least three distinct functions can now be identified: bend initiation, maintenance of the angle of propagating bends, and generation of power to overcome viscous resistances. Only the last of these three functions is an outer arm dynein function.

Animals↗
PubMed1994

Sperm chemotaxis: egg peptides control cytosolic calcium to regulate flagellar responses.

Fragmentary evidence indicates that intracellular [Ca2+] (Cai) mediates sperm chemotaxis. However, neither correlations of swimming responses to chemoattractant-induced alterations of Cai nor explanations of how chemoattractant gradients control Cai exist. Here Cai increases produced by the egg peptide speract-not previously known to cause flagellar responses--were prolonged by treatment with 3-isobutyl-1-methylxanthine (IBMX). Flagellar waveform asymmetry then increased 40% and swimming paths became tightly circular. Moreover, both responses required external Ca2+ (as does sperm chemotaxis to eggs and egg products). Cai increases by the established chemotactic peptide resact also required external Ca2+ and were enhanced by IBMX. Therefore, diverse egg peptides may use fundamentally similar mechanisms to control Cai and thereby swimming behavior in chemotaxis. Repetitive increasing additions of speract produced adaptive membrane potential and Cai responses indicating that sperm can detect increasing gradients of egg peptide over a broad concentration range. We offer a model in which shallow or decreasing gradients elevate Cai and redirect swimming paths but sufficiently steep gradients keep Cai low and swimming linear until the egg is reached. A negative-feedback loop, initiated by cGMP-mediated activation of sperm K+ channels and terminated by subsequent inactivation of guanylyl cyclase, may coordinate gradient detection with control of Cai. Continued stimulation of more receptors by steeply increasing gradients of egg peptide thus maintains membrane hyperpolarization and suppresses Ca2+ entry and Cai elevation. The molecular basis for chemotaxis therefore is explained as translation of the spatial gradient of peptide concentration into changes in K+ channel activity in the time domain.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

Microtubule sliding in reduced-amplitude bending waves of Ciona sperm flagella: resolution of metachronous and synchronous sliding components of stable bending waves.

Microtubule sliding associated with the bending of reactivated flagella of demembranated spermatozoa of the tunicate, Ciona, has been analyzed using a descriptive model that permits quantitation of metachronous and synchronous components of sliding. Reduced-amplitude bending waves, obtained by addition of increased salt (K acetate), lithium, or vanadate to the reactivation solutions, have been examined. Increased K acetate can decrease bend angle by as much as 70% with little change in frequency. In all cases, a decrease in the amplitude, or bend angle, of propagated bends is measured as a decrease in the metachronous component of sliding and is associated with a reduction in the growth of new bends after they begin to propagate during the second half-cycle of bend development. At higher K acetate concentrations, bend growth during the second half-cycle of bend development is very strongly reduced and may even become negative. A disparity between the rates of bend growth in the first and second half-cycles of bend development corresponds to a large amount of synchronous sliding in the distal portion of the flagellum. When the synchronous sliding component is large, the sliding velocity in a propagating bend decreases to near-0 values and may even reverse its direction as the bend propagates through the mid-region of the flagellum. Since these large perturbations of sliding velocity do not interfere with regular propagation of bends with nearly constant bend angle, the bend propagation mechanism cannot operate by metachronous control of the velocity of sliding, and is unlikely to operate by local monitoring of either the amount or velocity of sliding. These observations therefore argue against models in which active sliding is regulated by shear or sliding velocity, and make curvature-controlled models relatively more attractive. In many cases, a reduction in sliding during bend initiation (the first half-cycle of development of new bends) also contributes to the decreased amplitude of propagated bends. These changes in bend initiation are similar in both full-length flagella and in flagella shortened by breakage. The amount of sliding that occurs during bend initiation is relatively independent of the distribution of sliding between metachronous and synchronous components in the distal part of the flagellum. These observations therefore provide additional evidence that bend initiation and bend propagation are independent and separable processes.

Acetates↗
PubMed1991

Calcium sensors in sea urchin sperm flagella.

The asymmetry of ATP-reactivated flagellar bending waves of Triton-demembrated sea urchin spermatozoa has been measured over a range of free Ca2+ ion concentrations from 10(-9) to 10(-4) M. Detailed examination of the gradual response of asymmetry to Ca2+ ion concentration over this wide range indicates the presence of two Ca2+ sensors. A high-affinity sensor operates at Ca2+ concentrations near 10(-7.5) M. A lower-affinity sensor operates at Ca2+ concentrations above 10(-6) M, in the typical range for calmodulin-mediated responses. Incubation of demembranated sperm flagella at high Ca2+ concentrations to release calmodulin is required to enable these Ca2+ responses to be observed. This treatment also causes a decrease in the apparent affinity of the flagella for calmodulin, as determined by measuring the increase in asymmetry in response to addition of exogenous calmodulin at low Ca2+ concentration.

Animals↗
PubMed1991

Microtubule sliding in swimming sperm flagella: direct and indirect measurements on sea urchin and tunicate spermatozoa.

Direct measurements of microtubule sliding in the flagella of actively swimming, demembranated, spermatozoa have been made using submicron diameter gold beads as markers on the exposed outer doublet microtubules. With spermatozoa of the tunicate, Ciona, these measurements confirm values of sliding calculated indirectly by measuring angles relative to the axis of the sperm head. Both methods of measurement show a nonuniform amplitude of oscillatory sliding along the length of the flagellum, providing direct evidence that "oscillatory synchronous sliding" can be occurring in the flagellum, in addition to the metachronous sliding that is necessary to propagate a bending wave. Propagation of constant amplitude bends is not accomplished by propagation of a wave of oscillatory sliding of constant amplitude, and therefore appears to require a mechanism for monitoring and controlling the bend angle as bends propagate. With sea urchin spermatozoa, the direct measurements of sliding do not agree with the values calculated by measuring angles relative to the head axis. The oscillation in angular orientation of the sea urchin sperm head as it swims appears to be accommodated by flexure at the head-flagellum junction and does not correspond to oscillation in orientation of the basal end of the flagellum. Consequently, indirect calculations of sliding based on angles measured relative to the longitudinal axis of the sperm head can be seriously inaccurate in this species.

Animals↗
PubMed1991

Activation of Ciona sperm motility: phosphorylation of dynein polypeptides and effects of a tyrosine kinase inhibitor.

A high molecular mass dynein ATPase polypeptide and a 18-20 kDa dynein light chain of Ciona sperm flagella are phosphorylated during in vivo activation of motility or in vitro activation of motility by incubation with cyclic AMP. A similar level of phosphorylation of these proteins is obtained by incubation of washed, demembranated spermatozoa with catalytic subunit of cyclic AMP-dependent protein kinase, under conditions where there is no activation of motility until a supernatant component is added. Therefore, phosphorylation of these dynein polypeptides is not sufficient for activation of motility. Activation of motility in vitro by incubation with cyclic AMP can be completely inhibited by a random copolymer of glutamate and tyrosine that inhibits tyrosine kinase activity. Under these conditions, much of the protein phosphorylation associated with activation of motility is also inhibited. These new results suggest that regulation of motility of these spermatozoa may involve a multicomponent kinase cascade rather than a simple phosphorylation of a protein 'switch' by the cyclic AMP-dependent kinase. A 53 kDa axonemal phosphoprotein band, identified as band M1, shows the strongest correlation with activation of motility in these experiments.

Animals↗
PubMed1990

Computerized analysis of flagellar motility by digitization and fitting of film images with straight segments of equal length.

Methods are described for computerized analysis of digitized images obtained by scanning photomicrographs of swimming sperm flagella. After storing a series of image frames in computer memory, the entire series is analyzed automatically. For each sperm image, the sperm head is located to obtain a starting point for analysis of the flagellum. This location is obtained by minimizing image intensity along a model of the sperm head outline. The flagellum is modelled by a series of straight segments of equal length: 0.5 or 1 micron. The angles between these segments are adjusted to give minimum image intensity along the line of the model as well as minimizing smoothing functions. Extensions to analyze a series of images in each frame, and to measure the positions of beads attached to the flagellar microtubules, are also described.

Animals↗
PubMed1989

Direct measurements of sliding between outer doublet microtubules in swimming sperm flagella.

The relative motion of 40-nanometer gold beads bound to the exposed outer doublet microtubules of demembranated sea urchin sperm flagella has been observed and photographed during adenosine triphosphate (ATP)-reactivated swimming. This direct demonstration and measure of sliding displacements between outer doublet microtubules in actively bending flagella verifies the original sliding microtubule model for ciliary bending that was established by electron microscopy of fixed cilia and provides a new, functional measure for the diameter of the flagellar axoneme of 132 +/- 8 nanometers.

Adenosine Triphosphate↗
PubMed1989

2-Chloro adenosine triphosphate as substrate for sea urchin axonemal movement.

The 2-substituted ATP analog 2-Chloro ATP was tested for its capacity to support axonemal movement. The movement of sea urchin axonemes reactivated with 2-Cl ATP appeared very similar to that with ATP. Detailed waveform analysis indicated that bend angle and shear amplitude were not significantly different for ATP and 2-Cl ATP. Although wavelength differs at particular nucleotide concentrations, if normalized to the beat frequency, it is similar for ATP and 2-Cl ATP. The main difference in the movement with the two analogs was seen in beat frequency and sliding velocity. The Vmax for beat frequency and mean sliding velocity was lower for 2-Cl ATP. The apparent Km for beat frequency and sliding velocity was much lower for 2-Cl ATP. The ratio of these two effects, that is, (Vmax/Km) is higher for 2-Cl ATP. Thus 2-Cl ATP is a good substrate for axonemal movement. The significantly lower Km of 2-Cl ATP was also demonstrated by its ability to support oscillatory motion at concentrations below that for ATP. The observations identify the structures and conformation of substrate necessary to support axonemal movement.

Adenosine Triphosphate↗