Date of Award

2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Judith Van Houten

Abstract

Paramecium ciliary basal bodies align in straight rows from posterior to anterior. Each basal body is connected to three rootlets ((Post Ciliary Rootlet (PCR), Transverse Rootlet (TR) and Striated Rootlet (SR)). The SR, the longest, projects from the basal body toward the anterior past several more anterior basal bodies. The depletion of Meckelin (MKS3) misaligns SRs, disorganizes basal body rows and makes the SRs appear ragged and serpentine. In this study we clarify the composition of the Paramecium ciliary basal body’s SR and demonstrate that the SR plays a critical role in creating the orderly array of basal bodies in rows that run from pole to pole of the cell, likely through the interactions with centrins and other cytoskeletal elements underlying the cell surface. Here in this study we first report the reciprocal relationship between the SR and centrin related infraciliary lattice (ICL) protein that can dictate the cell surface morphology.

The SR of Chlamydomonas is the best studied. Using the single SR Chlamydomonas gene SF-assemblin to search in Paramecium DB, we found thirty Paramecium genes in thirteen Paralog Groups. Proteins from 13 paralog groups were confirmed to be in the SR structure using immunofluorescence. LC-MS/MS analyses of density fractions from SRs isolation show all thirty SR members are within the same density fraction. We further categorized all 30 SR genes in five Structural Groups based on their ability to form coiled coil domain and evaluate the function of all five Structural Group using RNA interference (RNAi). Silencing the transcripts of the any of the Structural Group showed misaligned basal body rows and the disordered organization of the SRs with abnormal appearance of SRs all over the cell surface. Silencing of Paralog Group showed normal phenotype except for the two Paralog Group (Paralog Group 1 or Paralog Group 7) which themselves constitute Structural Group individually. Isolated SRs from the control or Paralog Group depleted cells show a characteristic striation pattern that includes characteristic major and minor striations. Isolated SRs from any of the Structural Group depleted cells demonstrate abnormal shapes and striation periodicity. There is a correlation between the SR Structural Group RNAi surface misalignment phenotype and the isolated SR Structural Group RNAi phenotype for shape and periodicity of the SR. Strikingly our study of SR clearly demonstrates the role of SRs in shaping the other cytoskeleton structures of the cell cortex e.g., ICL, epiplasm territory and cortical unit territory.

In another follow up study of MKS3 (Picariello et al., 2014), we depleted the transcripts of MKS5 gene in Paramecium tetraurelia. Depletion of MKS5 transcripts in Paramecium causes cilia loss all over the cell surface. Unlike MKS3 depletion, MKS5 depletion does not affect the straight basal body rows and the ordered organization of SRs. Moreover, data presented in this study clearly demonstrates depletion of MKS5 transcripts somehow affect the localization of another transition zone protein, B9D2.

It appears when lacking any of the SR Structural Group, the rest fail to interact properly with each other to maintain the SRs structure and directionality toward the anterior. As a result, abnormal SRs appear to lose the interaction with other cytoskeleton structures such as ICL network complex, which eventually results in misaligned basal body rows and altered swimming behavior. From the data presented in this study it is reasonable to postulate ICL1e subfamily and SRs are in a reciprocal relationship to maintain the straight basal body rows and the highly ordered organization of the SRs all over the cell surface.

Language

en

Number of Pages

220 p.

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