Measurement of the force and torque produced in the calcium response of reactivated rat sperm flagella.

Rat sperm that are demembranated with Triton X-100 and reactivated with Mg-ATP show a strong mechanical response to the presence of free calcium ion. At pCa < 4, the midpiece region of the flagellum develops a strong and sustained curvature that gives the cell the overall appearance of a fishhook [Lindemann and Goltz, 1988: Cell Motil. Cytoskeleton 10:420-431]. In the present study, the force and torque that maintain the calcium-induced hook have been examined quantitatively. In addition, full-length and shortened flagella were manipulated to evaluate the plasticity of the hooks and determined the critical length necessary for maintaining the curvature. The hooks were found to be highly resilient, returning to their original configuration (>95%) after being straightened and released. The results from manipulating the shortened flagella suggest that the force holding the hook in the curved configuration is generated in the basal 60 microm of the flagellum. The force required to straighten the calcium-induced hooks was measured with force-calibrated glass microprobes, and the bending torque was calculated from the measured force. The force and torque required to straighten the flagellum were found to be proportional to the change in curvature of the hooked region of the flagellum, suggesting an elastic-like behavior. The average torque to open the hooks to a straight position was 2.6 (+/-1.4) x 10(-7) dyne x cm (2.6 x 10(-14) N x m) and the apparent stiffness was 4.3 (+/-1.3) x 10(-10) dyne x cm(2) (4.3 x 10(-19) N x m(2)). The stiffness of the hook was determined to be approximately one quarter the rigor stiffness of a rat sperm flagellum measured under comparable conditions.

Measurement of the force produced by an intact bull sperm flagellum in isometric arrest and estimation of the dynein stall force.

The force generated by a detergent-extracted reactivated bull sperm flagellum during an isometric stall was measured with a force-calibrated glass microprobe. The average isometric stall force from 48 individual measurements was 2.5 +/- 0.7 x 10(-5) dyne (2.5 +/- 0.7 x 10(-10) N). The force measurements were obtained by positioning a calibrated microprobe in the beat path of sperm cells that were stuck by their heads to a glass microscope slide. The average position of the contact point of the flagellum with the probe was 15 microm from the head-tail junction. This average lever arm length multiplied by the measured force yields an estimate of the active bending moment (torque) of 3.9 x 10(-8) dyne x cm (3.9 x 10(-15) N x m). The force was sustained and was for the most part uniform, despite the fact that the flagellum beyond the point of contact with the probe usually continued beating. It appears that the dynein motors in the basal portion of the flagellum continue to pull in an isometric stall for as long as the motion of the flagellum is blocked. If dynein motors in the flagellum distal to the contact point with the probe were contributing force to the displacement of the probe, then the flagellar segment immediately past the point of contact would have to show a net curvature in the direction of the probe displacement. No such curvature bias was observed in the R-bend arrests, and only a small positive curvature bias was measured in the P-bend arrests. Our analysis of the data suggests that more than 90% of the sustained force component is generated by the part of the flagellum between the probe and the flagellar base. Based on this premise, the isometric stall force per dynein head is estimated to be 5.0 x 10(-7) dyne (5 pN). This equals approximately 1.0 x 10(-6) dyne (10 pN) per intact dynein arm. These values are close to the isometric stall force of isolated dynein. This suggests that all of the dynein heads between the base and the probe, on the active side of the axoneme, are contributing to the force exerted against the probe.

Flagellar arrest behavior predicted by the Geometric Clutch model is confirmed experimentally by micromanipulation experiments on reactivated bull sperm.

The central tenet of the Geometric Clutch hypothesis of flagellar beating is that the internal force transverse to the outer doublets (t-force) mediates the initiation and termination of episodes of dynein engagement. Therefore, if the development of an adequate t-force is prevented, then the dynein-switching necessary to complete a cycle of beating should fail. The dominant component of the t-force is the product of the longitudinal force on each outer doublet multiplied by the local curvature of the flagellum. In the present study, two separate strategies, blocking and clipping, were employed to limit the development of the t-force in Triton X-100 extracted bull sperm models. The blocking strategy used a bent glass microprobe to restrict the flagellum during a beat, preventing the development of curvature in the basal portion of the flagellum. The clipping strategy was designed to shorten the flagellum by clipping off distal segments of the flagellum with a glass microprobe. This limits the number of dyneins that can contribute to bending and consequently reduces the longitudinal force on the doublets. The blocking and clipping strategies both produced an arrest of the beat cycle consistent with predictions based on the Geometric Clutch hypothesis. Direct comparison of experimentally produced arrest behavior to the behavior of the Geometric Clutch computer model of a bull sperm yielded similar arrest patterns. The computer model duplicated the observed behavior using reasonable values for dynein force and flagellar stiffness. The experimental data derived from both blocking and clipping experiments are fully compatible with the Geometric Clutch hypothesis.

Failure of highly immunogenic filarial proteins to provide host-protective immunity.

In areas that are endemic for lymphatic filariasis, there are individuals who are parasite free and who appear not to have experienced symptoms attributable to filarial infection. These "putatively immune" individuals may recognize immunogens that could be important in host protection. We have immunoscreened expression libraries expressing epitopes encoded by filarial open reading frames and have identified three antigens that are differentially recognized by the two polar clinical groups-endemic normals and asymptomatic microfilaremics. Pre-immunization of susceptible hosts (Meriones unguiculatus) with these antigens revealed that none was able to elicit consistent host protective immunity. Our data are consistent with Waksman's conjecture that highly immunogenic antigens of parasite origin may be inappropriate candidates for prophylactic immunization.

Localization of paramyosin, myosin, and a heat shock protein 70 in larval and adult Brugia malayi.

Recombinant filarial proteins are of interest as potentially protective immunogens for lymphatic filariasis. We have previously identified paramyosin, myosin, and a heat shock protein 70 (HSP) 70 as prominent immunogens in individuals residing in an area endemic for lymphatic filariasis. Our goal in the present work was to identify the Brugia malayi tissues that contain these proteins. Polyclonal rabbit antisera with high levels of immunoglobulins to each of these proteins were prepared for use in indirect immunofluorescence microscopy studies of third-and fourth-stage larvae (L3's and L4's) and adult worms. Myosin and paramyosin were found within the longitudinal somatic musculature in all of these life stages. In L4's and adult worms, myosin and paramyosin were also detected within the walls of the reproductive and alimentary tracts of male and female worms. HSP 70 was evident within the somatic musculature, hypodermis, lateral chords, alimentary tract, and reproductive structures in L4's and adult worms. HSP 70 was not detected in sections of freshly obtained L3's. However, L3's cultured at 37 C for 24 hr before fixation demonstrated a classic heat shock response. In these larvae, intracellular HSP 70 was observed in all tissues. None of the antigens studied appeared to be located on cuticular surfaces.

Infectivity and normal development of third stage Brugia malayi maintained in vitro.

Shipment of infective-stage filarial larvae (L3s) usually has been accomplished by transporting living infected vectors or L3s cryopreserved in liquid nitrogen. Our objective was to find culture conditions for transporting L3s that would promote survival of Brugia malayi larvae without altering their capacity to infect susceptible animals. In preliminary studies we observed that Ham's nutrient mixture F-12, with antibiotics and 1% fetal calf serum, could support L3s without apparent development for at least 10 days. In order to evaluate the effect of culture temperatures on infectivity, fresh L3s were divided into groups that were either immediately injected into jirds (infectivity control) or incubated for 24, 48, or 120 hr in tightly sealed tubes maintained horizontally at either 0 C, 20 C, or 37 C, before they were injected into jirds. Necropsies were performed on the jirds 120-130 days after injection to recover and count adult worms. Levels of microfilaremia were also determined. We found that L3s held overnight at 0 C, although apparently viable, were unable to survive in jirds. However, larvae kept at 20 C and 37 C produced patent infections with adult worms in normal locations even after 120 hr of in vitro cultivation. There was no statistical difference in mean worm recovery or size of worms from jirds infected with freshly harvested L3s and jirds injected with larvae that were maintained overnight at 20 C or 37 C. When cultured L3s were shipped from Michigan to Connecticut by overnight air courier, along with infected living mosquitos, the L3s appeared to be 99% viable upon arrival. L3s shipped in F-12 produced patent infections in C.B.-17 scid/scid mice with worm recoveries comparable to those observed in mice injected with L3s freshly obtained from shipped mosquitos.