Date of Award

10-1978

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Supervisor

Dr. A.D. Dingle

Abstract

Naegleria gruberi amoebae differentiate rapidly and synchronously to flagellates, assembling a complete flagellar apparatus de novo within two hours. The flagellar apparaturs consists of two flagella, their associated basal bodies and a flagellar rootlet. This study is directed towards an understanding of the development of the flagellar rootlet. The rootlet has been isolated by differential centrifugation and the major rootlet protein has been identified by solubilization of the organelle in urea, high salt, detergents or extremes in pH.

During the amoeba to flagellate differentiation at least 62% of the major rootlet protein assembled is synthesized de novo as measured by means of an isotope dilution experiment. Most likely the major rootlet protein is completely synthesized de novo since it is absent in amoebae but present in developing flagellates prior to the appearance of rootlets. This protein, whose subunit molecular weight is 170,000 daltons, has been purified by elution from polyacrylamide preparative gels. The sequence of rootlet development has been eluciated by indirect immunofluorescence using a specific antibody directed against the purified major rootlet protein. Rootlets, which are first detected in developing flagellates 70 min after the initiation of differentiation, reach their maximum length of 13 μm within 40 min. During the differentiation, the appearance of rootlets in the population follows that of flagella, but is delayed by about 5 min. When flagellates revert to amoebae, the rootlets disappear from cells shortly after the flagella are lost, and they reappear just minutes after the flagella become visible in redifferentiating flagellates.

The controls which govern the number of organelles assembled during differentiation are not absolute and cells may assemble three, four or occasionally more flagella. Furthermore, the controls of organelle number may be deranged by high temperature shocks applied at precisely defined intervals during the differentiation such that the resulting flagellated populations may develop nearly three times the normal number of basal bodies and flagella. In these multiflagellated popultions the increase in the mean number of rootlets per cell is proportional to the increase in the mean number of flagella such that the two flagella to one rootlet relationship of a typical (biflagellate) flagellar apparatus is mainteained. These results suggest that the temperature effect which leads to uncontolled basal body and flagellum assembly simltaneously deranges whatever controls the number of rootlets assembled.

As the number of flagella and rootlets per cell increases, the mean length of the organelles decreases. These do not counter-balance, however, and there is a net increase in the total length of organelle assembled. Measurements of the total amount of the major rootlet protein in control and temperature-shocked populations show no increase in the multiflagellated cells. Rather, there appears to be an increase in utilization of the available major rootlet protein to provide for the increased rootlet assembled.

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