In vivo Analysis of the Role of FtsZ1 and FtsZ2 Proteins in Chloroplast Division in Arabidopsis thaliana

Chloroplasts divide by a constrictive fission process that is regulated by FtsZ proteins. Given the importance of photosynthesis and chloroplasts in general, it is important to understand the mechanisms and molecular biology of chloroplast division. An FtsZ gene is known to be of prokaryotic origin...

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Main Author: Johnson, Carol
Other Authors: Holzenburg, Andreas
Format: Others
Language:en_US
Published: 2012
Subjects:
Online Access:http://hdl.handle.net/1969.1/ETD-TAMU-2012-05-10930
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spelling ndltd-tamu.edu-oai-repository.tamu.edu-1969.1-ETD-TAMU-2012-05-109302013-01-08T10:44:05ZIn vivo Analysis of the Role of FtsZ1 and FtsZ2 Proteins in Chloroplast Division in Arabidopsis thalianaJohnson, CarolArabidopsis thalianaFtsZ1FtsZ2correlative microscopychloroplast divisionChloroplasts divide by a constrictive fission process that is regulated by FtsZ proteins. Given the importance of photosynthesis and chloroplasts in general, it is important to understand the mechanisms and molecular biology of chloroplast division. An FtsZ gene is known to be of prokaryotic origin and to have been transferred from a symbiont's genome to host genome via lateral transfer. Subsequent duplication of the initial FtsZ gene gave rise to the FtsZ1 and FtsZ2 genes and protein families in eukaryotes. These proteins co-localize mid-chloroplast to form the Z-ring. Z-ring assembly initiates chloroplast division, and it serves as a scaffold for other chloroplast division proteins. Little is known, however, about the FtsZ protein subunit turnover within the Z-ring, the effects of accessory proteins on Z-ring turnover assemblies, as well as the in vivo ultrastructure of the Z-ring in plants. To investigate the Arabidopsis thaliana FtsZ subunit turnover rate within the Z-ring, a section of the Z-ring in the chloroplasts of living plants expressing fluorescently tagged FtsZ1 or FtsZ2 proteins was photobleached and the recovery rate was monitored. The results show that the fluorescence recovery half times for the FtsZ1 and FtsZ2 proteins are 117s and 325s, respectively. This is significant as these data mirror their differences in GTP hydrolysis rates. To elucidate in vivo structure and ultrastructure of the Z-ring, a protocol was established that maintained fluorescence during high pressure freezing, freeze substitution and low temperature embedding. Afterwards, a correlative microscopy approach was employed to visualize and identify fluorescently labeled puncta, circular structures, at the light microscopy level. These puncta were further resolved as mini-rings using optical microscopy eXperimental (OMX) superresolution microscopy. Electron microscopy (EM) analysis imaged mini-rings and filament assemblies comprised of dense subunits. Electron tomography (ET) showed mini-rings composed of protofilaments.Holzenburg, Andreas2012-07-16T15:58:07Z2012-07-16T20:30:25Z2012-07-16T15:58:07Z2012-052012-07-16May 2012thesistextapplication/pdfhttp://hdl.handle.net/1969.1/ETD-TAMU-2012-05-10930en_US
collection NDLTD
language en_US
format Others
sources NDLTD
topic Arabidopsis thaliana
FtsZ1
FtsZ2
correlative microscopy
chloroplast division
spellingShingle Arabidopsis thaliana
FtsZ1
FtsZ2
correlative microscopy
chloroplast division
Johnson, Carol
In vivo Analysis of the Role of FtsZ1 and FtsZ2 Proteins in Chloroplast Division in Arabidopsis thaliana
description Chloroplasts divide by a constrictive fission process that is regulated by FtsZ proteins. Given the importance of photosynthesis and chloroplasts in general, it is important to understand the mechanisms and molecular biology of chloroplast division. An FtsZ gene is known to be of prokaryotic origin and to have been transferred from a symbiont's genome to host genome via lateral transfer. Subsequent duplication of the initial FtsZ gene gave rise to the FtsZ1 and FtsZ2 genes and protein families in eukaryotes. These proteins co-localize mid-chloroplast to form the Z-ring. Z-ring assembly initiates chloroplast division, and it serves as a scaffold for other chloroplast division proteins. Little is known, however, about the FtsZ protein subunit turnover within the Z-ring, the effects of accessory proteins on Z-ring turnover assemblies, as well as the in vivo ultrastructure of the Z-ring in plants. To investigate the Arabidopsis thaliana FtsZ subunit turnover rate within the Z-ring, a section of the Z-ring in the chloroplasts of living plants expressing fluorescently tagged FtsZ1 or FtsZ2 proteins was photobleached and the recovery rate was monitored. The results show that the fluorescence recovery half times for the FtsZ1 and FtsZ2 proteins are 117s and 325s, respectively. This is significant as these data mirror their differences in GTP hydrolysis rates. To elucidate in vivo structure and ultrastructure of the Z-ring, a protocol was established that maintained fluorescence during high pressure freezing, freeze substitution and low temperature embedding. Afterwards, a correlative microscopy approach was employed to visualize and identify fluorescently labeled puncta, circular structures, at the light microscopy level. These puncta were further resolved as mini-rings using optical microscopy eXperimental (OMX) superresolution microscopy. Electron microscopy (EM) analysis imaged mini-rings and filament assemblies comprised of dense subunits. Electron tomography (ET) showed mini-rings composed of protofilaments.
author2 Holzenburg, Andreas
author_facet Holzenburg, Andreas
Johnson, Carol
author Johnson, Carol
author_sort Johnson, Carol
title In vivo Analysis of the Role of FtsZ1 and FtsZ2 Proteins in Chloroplast Division in Arabidopsis thaliana
title_short In vivo Analysis of the Role of FtsZ1 and FtsZ2 Proteins in Chloroplast Division in Arabidopsis thaliana
title_full In vivo Analysis of the Role of FtsZ1 and FtsZ2 Proteins in Chloroplast Division in Arabidopsis thaliana
title_fullStr In vivo Analysis of the Role of FtsZ1 and FtsZ2 Proteins in Chloroplast Division in Arabidopsis thaliana
title_full_unstemmed In vivo Analysis of the Role of FtsZ1 and FtsZ2 Proteins in Chloroplast Division in Arabidopsis thaliana
title_sort in vivo analysis of the role of ftsz1 and ftsz2 proteins in chloroplast division in arabidopsis thaliana
publishDate 2012
url http://hdl.handle.net/1969.1/ETD-TAMU-2012-05-10930
work_keys_str_mv AT johnsoncarol invivoanalysisoftheroleofftsz1andftsz2proteinsinchloroplastdivisioninarabidopsisthaliana
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