UVM Theses and Dissertations
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Print
Author:
Svinkina, Tatyana
Title:
Dept./Program:
Biology
Degree:
MS
Abstract:
Target of rapamycin (TOR) is a highly conserved serine-threonine kinase. In eukaryotes it exists in two complexes. TORC1 controls cellular processes by responding to the presence of nutrients and growth factors in the environment. Growth factor insulin regulates TORC I through the PI3K (phosphoinositol-3-kinase)/Akt/TSC (tuberous sclerosis) signaling pathway. Amino acids regulate TORCI through small G proteins Rag GTPases. TOR activation can be regulated by the ciliary signaling. Cilia are hair-like projections with microtubule based cytoskeleton. Ciliary assembly depends on intraflagellar transport (1FT) that is powered by anterograde kinesin II and retrograde dynein 2 motors. Ciliary membrane contains voltage sensitive and calcium dependent ion channels, which function in ciliary mediated signaling.
TOR over expression leads to ciliary elongation, but direct mechanism is unknown. TOR possibly regulates cilia through Glycogen Synthase Kinase 3 (GSK3). GSK3 is a serine-threonine kinase that regulates microtubule assembly/disassembly and many other processes. GSK3 has been localized to the cilia of the murine kidney tissue, and to the flagella of Chlamydomonas reinhardtii. GSK3 might work together with cAMP activated protein kinase A (PKA) in the regulation of ciliary length and assembly. Here we investigate TOR signaling pathway orthologs and GSK3 effect on cilia in the unicellular ciliate protozoan Paramecium tetraurelia (P. tetraurelia).
Using BLAST (Basic Local Alignment Search Tool) we identified orthologs of mammalian TOR, LST8 (TORC1 member), Akt, Rheb, Rag AlB, Rag C/D, SNAT2 (amino acid transporter), Tap42 (TOR substrate), S6K (TOR substrate), PKA, and GSK3 in P.tetraurelia. We employed RNA interference (RNAi) and established that TOR, LST8, Rag A, Rag C, SNAT2, Tap42, S6K, and GSK3 depletion reduces cellular proliferation in P. tetraurelia. Using fluorescent microscopy we also determined that TOR, Rag C, and SNAT2 down-regulation arrests P. tetraurelia cells between Gland S stages of the cell cycle. TOR depletion had no visible effect on cilia, but test cells had significantly slower swimming speed when compated to controls. We propose that TOR might regulate expression and trafficking of ion channels to the cilia.
P. tetraurelia depends on the presence of these channels to respond to the extracellular environment and their absence will affect ciliary signaling and cell speed. GSK3 down-regulation produced round looking cells with short, sparse cilia. We created Flag-GSK3 construct and with immunofluorescence and Western blots confirmed GSK3 localization to the pellicle and cilia of P. tetraurelia. We also established that inhibition of PKA prevents ciliary re-growth in deciliated cells. Our results suggest that both GSK3 and PKA regulate ciliary assembly, but we have not established any link between PKA and GSK3. Possible explanation for the GSK3 depletion phenotype is that GSK3 phosphorylates KAP subunit ofthe kinesin II motor, and PKA could function as a priming kinase for GSK3 phosphorylation ofKAP. KAP phosphorylation would affect interaction between 1FT rafts and kinesin II and prevent cargo release required for ciliary assembly. We have not established if GSK3 and PKA function depends on the TOR signaling, but it would be interesting to determine the link in the future experiments. Our data provide interesting insights on the TOR and GSK3 function in P. tetraurelia and will hopefully promote further study ofthese two proteins in ciliates.
TOR over expression leads to ciliary elongation, but direct mechanism is unknown. TOR possibly regulates cilia through Glycogen Synthase Kinase 3 (GSK3). GSK3 is a serine-threonine kinase that regulates microtubule assembly/disassembly and many other processes. GSK3 has been localized to the cilia of the murine kidney tissue, and to the flagella of Chlamydomonas reinhardtii. GSK3 might work together with cAMP activated protein kinase A (PKA) in the regulation of ciliary length and assembly. Here we investigate TOR signaling pathway orthologs and GSK3 effect on cilia in the unicellular ciliate protozoan Paramecium tetraurelia (P. tetraurelia).
Using BLAST (Basic Local Alignment Search Tool) we identified orthologs of mammalian TOR, LST8 (TORC1 member), Akt, Rheb, Rag AlB, Rag C/D, SNAT2 (amino acid transporter), Tap42 (TOR substrate), S6K (TOR substrate), PKA, and GSK3 in P.tetraurelia. We employed RNA interference (RNAi) and established that TOR, LST8, Rag A, Rag C, SNAT2, Tap42, S6K, and GSK3 depletion reduces cellular proliferation in P. tetraurelia. Using fluorescent microscopy we also determined that TOR, Rag C, and SNAT2 down-regulation arrests P. tetraurelia cells between Gland S stages of the cell cycle. TOR depletion had no visible effect on cilia, but test cells had significantly slower swimming speed when compated to controls. We propose that TOR might regulate expression and trafficking of ion channels to the cilia.
P. tetraurelia depends on the presence of these channels to respond to the extracellular environment and their absence will affect ciliary signaling and cell speed. GSK3 down-regulation produced round looking cells with short, sparse cilia. We created Flag-GSK3 construct and with immunofluorescence and Western blots confirmed GSK3 localization to the pellicle and cilia of P. tetraurelia. We also established that inhibition of PKA prevents ciliary re-growth in deciliated cells. Our results suggest that both GSK3 and PKA regulate ciliary assembly, but we have not established any link between PKA and GSK3. Possible explanation for the GSK3 depletion phenotype is that GSK3 phosphorylates KAP subunit ofthe kinesin II motor, and PKA could function as a priming kinase for GSK3 phosphorylation ofKAP. KAP phosphorylation would affect interaction between 1FT rafts and kinesin II and prevent cargo release required for ciliary assembly. We have not established if GSK3 and PKA function depends on the TOR signaling, but it would be interesting to determine the link in the future experiments. Our data provide interesting insights on the TOR and GSK3 function in P. tetraurelia and will hopefully promote further study ofthese two proteins in ciliates.