Identification of interaction between septin3 and p60-katanin (KATNA1) proteins

p60-katanin, the small subunit of katanin protein, is an important protein regulating microtubule dynamics of mitotic cells during mitosis and neuronal branching in post-mitotic cells. The C-terminal AAA (ATPases Associated with diverse cellular Activities) domain containing ATPase activity carries microtubule severing function and causes formation of short microtubule pieces from long microtubules. Microtubule severing is an important cellular event which enables rapid re-organization of microtubule network during cell division in the cells. Besides, microtubule severing takes important roles in the formation and branching of neuronal processes such as axons and dendrites. Although p60-katanin is a protein widely worked on, neither its functioning mechanisms nor its regulatory partners are clearly determined. There are a few proteins identified so far that physically interact with p60-katanin. One of these proteins is the other subunit of katanin: p80-katanin. These two subunits exist as a heterodimer in cells. In our laboratory, p60-katanin interacting proteins were identified by Yeast Two Hybrid screening system. Human brain cDNA library was screened and twenty-two candidate proteins were identified. Septin3, present in pre-synaptic termini, having roles in synapse formation, has been chosen in this study. Septin 3 is a novel member of the Septin subfamily of GTPase domain proteins. The septin family consists of multiple genes and protein isoforms; in mammals, 13 septin genes encode for over 30 protein isoforms. Mostly all Septin proteins are expressed in many tissues, but Septin3 appears to be primarily expressed in brain. Septins are abundant in the central nervous system and associated with many neurological diseases such as Parkinson's, Alzheimer's, schizophrenia, and hereditary neuralgic amyotrophy. In this study, in order to analyze the interaction between p60-katanin and Septin3, co-immunoprecipitation assays were performed using rat brain lysate, and it was found that p60-katanin interacts with Septin3. Then, immunocytochemistry (ICC) was performed to monitor localization of endogenous Septin3 and p60-katanin proteins. Based on the ICC results, Septin3 mainly localizes along axons and p60-katanin mostly resides in cell bodies. These two proteins have been observed as co-localized along axons and cell body. In order to observe filament formation of Septin3, wild-type Septin3 construct was overexpressed in RFL6 cells, for better visualization due to its big and flat nature, and they were subjected to ICC. Wild-type Septin3 protein formed form brush-like filaments. Since the GTPase activity of septin proteins is important for filament formation, two critical amino acids, Lys208 and Arg280, for the GTPase activity of Septin3 were mutated both separately and together by site-directed mutagenesis. A mutation on Arg280 showed greater inhibitory effect on the GTPase activity of Septin3 than a mutation on Lys208. The lowest GTPase activity among Septin3 mutants were detected when both amino acids were mutated. Double mutant Septin3 construct was overexpressed in RFL6 cells and subjected to ICC. It was found that double mutant Septin3 protein was observed as being punctiform and accumulating on the cell membranes where they co-localized with microtubules. Septin3 wild-type and double mutant constructs were overexpressed in RFL6 cells together with p60-katanin and the cells were subjected to ICC. Wild-type Septin3 and wild-type p60-katanin co-transfected cells were observed to be smaller and to have disorganized microtubule structure than the cells expressing only Septin3. In addition, short Septin3 filaments were observed to be localized around p60-katanin proteins, whereas brush-like Septin3 filaments were observed in the cells expressing only Septin3. Double mutant Septin3 and wild-type p60-katanin co-transfected RFL6 cells have been observed to lack the ability to form septin filaments and they were overlaying with microtubules which seemed to have lost their regular meshwork. In order to identify the interaction domains of p60-katanin and Septin3, deletion constructs of p60-katanin and Septin3 were generated by cloning rat p60-katanin fragments into FLAG-tagged mammalian expression vector and by cloning rat Septin3 fragments into Myc/His-tagged mammalian expression vector. Then the constructs were overexpressed in human colon carcinoma cell line, due to its easily transfectable nature, and the total protein were subjected to co-immunoprecipitation (co-IP) assays. According to co-IP result, p60-katanin strongly binds to Septin3 via when it has ATPase domain at C-terminal end. In addition, Septin3 binds to p60-katanin through its GTPase domain in between 67th and 217th amino acids.