Microtubules are indispensable dynamic structures that contribute to many essential biological functions including cell division, intracellular transport and maintenance of cell shape or cell motility. Assembly of the native αβ-tubulin heterodimer, the subunit that polymerizes to form microtubules, requires participation of chaperonin CCT. The cytosolic chaperonin CCT is a heterooligomeric complex of about 900 kDa present in all eukaryotic cells. Increasing number of proteins has been shown to interact with CCT. One of them is phosducin-like protein (PhLP) which may play regulatory function in this complex. However, the potential role of PhLP in tubulin folding mechanism has not been explicated so far.

It has been shown in Tetrahymena thermophila ciliates that CCT protein is associated with microtubule structures and is involved in cilia biogenesis. These cells covered with thousands of cilia can be relatively easy deciliated therefore they present excellent model for study putative function of PhLP as a regulator of CCT-mediated tubulin folding during cilia regeneration. For the above reasons the purpose of this study was to verify if PhLP present in these ciliates may be engaged in the control of the tubulin folding. To achieve this immunological methods, cell fractionation and protein expression were employed. For investigation of PhLP and CCT expression during cilia regeneration, samples of total protein extract were analyzed by Western blot using antibodies specifically recognizing PhLP and CCTε. These experiments showed that levels of PhLP and CCTε decreased significantly after deciliation and then expression of these proteins started to increase reaching after 60 min the higher levels than in control. Cilia after this period were almost fully regenerated. Localizations of CCTε and PhLP in control, deciliated and reciliating cells were accomplished by laser confocal microscopy and cell fractionation method. To attain this tested ciliates were fractionated in 5 parts: cytosol (Sm), microsomal (Pm), cilia (Cc), soluble (Str) and insoluble (Ptr) in TritonX-100 cortex fractions. The experiments evidenced that major amount of PhLP in control cells was localized mainly in Pm and Str fractions. After cilia removing PhLP was detected mostly in Str fraction and expression of this protein increased with time. CCTε in control cells was detected in Cc, Str and Pm cell fractions and after deciliation partial translocation of the CCTε to Sm fraction was observed. Immunocytochemical experiments evidenced that PhLP and CCTε were colocalized with β-tubulin in control and deciliated cells. Observed changes in localization of PhLP and CCTε and colocalization of these proteins with β-tubulin in control and deciliated cells can indicate that in Tetrahymena thermophila ciliate both proteins may be involved in process of tubulin folding and/or microtubule assembly.

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