Condensin and chromosome condensation in the ciliate Tetrahymena thermophila

Condensin is a conserved protein complex important for promoting condensation and proper segregation of chromosomes in both mitosis and meiosis, and it has been implicated in cancer and human genetic disorders. Studies of condensin in diverse eukaryotes have shown quite a bit of variability in the absolute requirement of condensin for chromosome condensation, and have identified a diverse array of other functions. To expand on this knowledge, we propose to study condensin in the ciliated protist Tetrahymena thermophila. Like other ciliates, Tetrahymena is bi-nucleate. Its germline nucleus is diploid and divides by mitosis and meiosis, but is transcriptionally silent. The transcriptionally active somatic nucleus is polyploid, and contains about 50 copies each of 200 minichromosomes. These small chromosomes do not notably condense and do not contain centromeres, and thus are distributed randomly to daughter nuclei during somatic nuclear division. A previous study on the condensin subunit SMC4 in Tetrahymena demonstrated that condensin was required for the amitotic division of the somatic nucleus, but its role in this process, and in mitosis and meiosis of the germline, remains elusive. We have identified multiple homologs of some of the condensin subunits in Tetrahymena. Preliminary tagging and localization of select subunits suggest that multiple condensin complexes are active in Tetrahymena, and raise the possibility that some complexes may have specific functions in different processes performed by the germline and somatic nuclei. Depletion of cohesin by RNA interference (RNAi) against the shared subunit SMC2 has shown that cohesin is required for segregation of chromosomes in meiosis and mitosis, as well as for MAC division. For the project, we will perform experiments using novel fluorescence imaging techniques in both live and fixed cells. Localization and expression dynamics of all tagged condensin subunits will be determined, and RNAi depletion of different subunits will be used to investigate their functions in chromosome segregation in the somatic and germline nuclei. The timing and extent of chromosome condensation in meiosis, and the contribution of condensin to this process, will be monitored by live cell imaging. Fluorescence in situ hybridization (FISH) will be employed to investigate condensation and chromosome organization in the somatic nucleus. The post-translational modifications of condensin subunits will be examined to learn more about the mechanism and regulation of condensin. By studying condensin in Tetrahymena, which is evolutionarily distant from other model organisms, we hope to gain insight into the conserved functions of condensin, as well as investigate aspects unique to the nuclear biology of this interesting model system.

The genetic material of the cell, DNA, and its surrounding proteins that make up the chromosomes, exist in a dispersed form in non-dividing cells. In order to pack the chromosomes into compact structures that can be easily divided, the cell employs a protein complex called condensin. There are several forms of condensin, and they have multiple roles in regulating diverse cellular processes, many of which are still poorly understood. Malfunctions of these proteins can cause various congenital diseases in humans, including cancer and infertility. However, it is difficult to study these diseases in humans, because in order to analyze the function of condensins it is necessary to inactivate the corresponding genes and observe the consequences of their loss. Therefore this is done in "model" organisms. Tetrahymena thermophila, a relative of the slipper animalcule, is evolutionarily very distantly related to humans. Therefore, if a cellular process is found to be similar, it may be considered as primordial and fundamental. We replaced condensin genes by non-functional copies and studied the resulting chromosomal defects by microscopy. Also, we tagged condensin proteins to follow their distribution at each stage in cell division and identify proteins that interact with condensins. We found that Tetrahymena has more versions of condensin than any other organism studied to date, and some of these versions have unexpected functions. Most importantly, daughter chromosomes were unable to separate during cell division in the absence of condensin. In higher organisms, various versions exist of a related protein complex, cohesin, which has functions in gene regulation and in the orderly segregation of chromosomes during cell division. However, in Tetrahymena cohesins play a minor role, and we speculate that this declining importance may be due to the takeover of some of its functions by condensins. Notably, the depletion of condensin also fundamentally altered chromosome arrangement in the polyploid somatic nucleus of Tetrahymena: multiple copies of homologous chromosomes tended to cluster instead of being dispersed in the nucleus. It is believed that the territorial organization of chromosomes within the nucleus contributes to the regulation of gene activities. Therefore, the mislocalization of chromosomes may be the cause of various clinical consequences in condensin-related diseases. Finally, we identified a novel condensin function: In Tetrahymena, some of the genetic material that is present in the germline is eliminated in the soma, i.e., the nuclei that are not passed on to the offspring during sexual reproduction. When one version of condensin was inactivated, this elimination did not occur. This result highlights the evolutionary flexibility of condensin in acquiring novel functions, and also points to the possibility of similar functions that have yet to be detected in other organisms.