Asai, David John (1980) Immunological approaches to flagellar movement. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-08232006-093449
This thesis summarizes attempts in our laboratory to use antibodies to study flagellar motility. After preliminary work on the effects of antibodies to intact dynein 1 and a tryptic fragment of dynein 1, a thorough study of antibodies against outer doublet tubulin has been made.
Both anti-dynein 1 and anti-fragment 1A inhibit movement-coupled ATP dephosphorylation and the movement of reactivated Strongylocentrotus purpuratus spermatozoa. Anti-fragment 1A, which also inhibits the ATPase activity of isolated dynein, inhibits the frequency and bend angle of the flagella. Anti-dynein 1 has a relatively smaller effect on beat frequency and is inhibitory of bend angle. These results suggest that binding to dynein is not, by itself, sufficient to strongly inhibit movement and that a more specific interference with ATPase activity produces more severe effects on movement.
Antibodies to tubulin were purified from rabbit sera by tubulin-affinity chromatography. Four induced anti-tubulins from immune sera and four spontaneous anti-tubulins from the corresponding preimmune sera were isolated and characterized. Both induced and spontaneous anti-tubulins are of the IgG class of antibody. Both are specific for tubulin when presented with a crude mixture of axonemal proteins. Only induced anti-tubulin precipitates tubulin in immunodiffusion assays. Both anti-tubulins as well as their monovalent Fab fragments bind to sea urchin axonemes in a radiobinding assay. Both stain demembranated sperm flagella in indirect immunofluorescence but only induced anti-tubulin decorates chick fibroblast cytoplasmic microtubules. I conclude that the affinity-purified antibodies are specific for tubulin and are able to bind sea urchin sperm flagella.
Induced anti-tubulins from four different immunizations all specifically reduce bend angle and symmetry of reactivated flagella without affecting beat frequency. At identical concentrations, spontaneous anti-tubulins, and immune and preimmune Fab fragments have no effect on any of the parameters of flagellar movement. The ATP-mediated disintegration of elastase-digested axonemes is not prevented by concentrations of induced anti-tubulin which are more than sufficient to paralyze reactivated spermatozoa, but the ATP concentration threshold for sliding is raised. Under similar conditions, anti-fragment 1A has been shown to completely inhibit microtubule sliding. Induced anti-tubulin behaves very similarly to CO2 in its movement-inhibitory activity.
These three antibodies all inhibit the bend angle of the reactivated flagella; they represent the first reports of amplitude inhibition by reagents known to be specific for the components responsible for active sliding. The specific bend angle inhibition and lack of effect on sliding by induced anti-tubulin is consistent with a microtubule conformational control of cross-bridge activity as a mechanism controlling the bending of the flagellum. The induced anti-tubulin effect is the first report of an antibody to tubulin having an inhibitory activity on microtubule-associated movement.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||9 August 1979|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||29 Aug 2006|
|Last Modified:||26 Dec 2012 02:58|
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