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Compounds that disrupt microtubule dynamics, such as colchicine, paclitaxel, or Vinca alkaloids, have been broadly used in biological studies and have found application in clinical anticancer medications. However, their main disadvantage is the lack of specificity towards cancerous cells, leading to severe side effects. In this paper, we report the first synthesis of 12 new visible light photoswitchable colchicine-based microtubule inhibitors AzoCols . Among the obtained compounds, two photoswitches showed light-dependent cytotoxicity in cancerous cell lines (HCT116 and MCF-7). The most promising compound displayed a nearly twofold increase in potency. Moreover, dissimilar inhibition of purified tubulin polymerisation in cell-free assay and light-dependent disruption of microtubule organisation visualised by immunofluorescence imaging sheds light on the mechanism of action as microtubule photoswitchable destabilisers. The presented results provide a foundation towards the synthesis and development of a novel class of photoswitchable colchicine-based microtubule polymerisation inhibitors.

Cancer is one of the most common causes of human death worldwide; thus, numerous therapies, including chemotherapy, have been and are being continuously developed. In cancer cells, an aberrant mitotic spindle-a microtubule-based structure necessary for the equal splitting of genetic material between daughter cells-leads to genetic instability, one of the hallmarks of cancer. Thus, the building block of microtubules, tubulin, which is a heterodimer formed from α- and β-tubulin proteins, is a useful target in anti-cancer research. The surface of tubulin forms several pockets, i.e., sites that can bind factors that affect microtubules' stability. Colchicine pockets accommodate agents that induce microtubule depolymerization and, in contrast to factors that bind to other tubulin pockets, overcome multi-drug resistance. Therefore, colchicine-pocket-binding agents are of interest as anti-cancer drugs. Among the various colchicine-site-binding compounds, stilbenoids and their derivatives have been extensively studied. Herein, we report systematic studies on the antiproliferative activity of selected stilbenes and oxepine derivatives against two cancer cell lines-HCT116 and MCF-7-and two normal cell lines-HEK293 and HDF-A. The results of molecular modeling, antiproliferative activity, and immunofluorescence analyses revealed that compounds 1a , 1c , 1d , 1i , 2i , 2j , and 3h were the most cytotoxic and acted by interacting with tubulin heterodimers, leading to the disruption of the microtubular cytoskeleton.

Primary ciliary dyskinesia (PCD) is a hereditary genetic disorder caused by the lack of motile cilia or the assembxly of dysfunctional ones. This rare human disease affects 1 out of 10,000-20,000 individuals and is caused by mutations in at least 50 genes. The past twenty years brought significant progress in the identification of PCD-causative genes and in our understanding of the connections between causative mutations and ciliary defects observed in affected individuals. These scientific advances have been achieved, among others, due to the extensive motile cilia-related research conducted using several model organisms, ranging from protists to mammals. These are unicellular organisms such as the green alga Chlamydomonas , the parasitic protist Trypanosoma , and free-living ciliates, Tetrahymena and Paramecium , the invertebrate Schmidtea , and vertebrates such as zebrafish, Xenopus , and mouse. Establishing such evolutionarily distant experimental models with different levels of cell or body complexity was possible because both basic motile cilia ultrastructure and protein composition are highly conserved throughout evolution. Here, we characterize model organisms commonly used to study PCD-related genes, highlight their pros and cons, and summarize experimental data collected using these models.

Motile cilia are ultrastructurally complex cell organelles with the ability to actively move. The highly conserved central apparatus of motile 9 × 2 + 2 cilia is composed of two microtubules and several large microtubule-bound projections, including the C1b/C1f supercomplex. The composition and function of C1b/C1f subunits has only recently started to emerge. We show that in the model ciliate Tetrahymena thermophila, C1b/C1f contains several evolutionarily conserved proteins: Spef2A, Cfap69, Cfap246/LRGUK, Adgb/androglobin, and a ciliate-specific protein Tt170/TTHERM_00205170. Deletion of genes encoding either Spef2A or Cfap69 led to a loss of the entire C1b projection and resulted in an abnormal vortex motion of cilia. Loss of either Cfap246 or Adgb caused only minor alterations in ciliary motility. Comparative analyses of wild-type and C1b-deficient mutant ciliomes revealed that the levels of subunits forming the adjacent C2b projection but not C1d projection are greatly reduced, indicating that C1b stabilizes C2b. Moreover, the levels of several IFT and BBS proteins, HSP70, and enzymes that catalyze the final steps of the glycolytic pathway: enolase ENO1 and pyruvate kinase PYK1, are also reduced in the C1b-less mutants.

Introduction: CacyBP/SIP is a multifunctional protein present in various mammalian tissues, among them in brain. Recently, it has been shown that CacyBP/SIP exhibits phosphatase activity towards ERK1/2 and p38 kinases., Objectives: The aim of our study was to analyze the localization and level of CacyBP/SIP and its substrates, phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated p38 (p-p38) kinases, in an intact and transected rat spinal cord., Methods: To achieve our goals we have performed Western blot/densitometric analysis and double immunofluorescence staining using rat spinal cord tissue, intact and after total transection at different time points., Results: We have observed a decrease in the level of CacyBP/SIP and an increase in the level of p-ERK1/2 and of p-p38 in fragments of the spinal cord excised 1 and 3 months after transection. Moreover, immunofluorescence staining has shown that CacyBP/SIP, p-ERK1/2 or p-p38 co-localized with a neuronal marker, NeuN, and with an oligodendrocyte marker, Olig2., Conclusion: The inverse correlation between CacyBP/SIP and p-ERK1/2 or p-p38 levels suggests that CacyBP/SIP may dephosphorylate p-ERK1/2 and p-p38 kinases and be involved in neural plasticity following spinal cord injury., Competing Interests: Declaration of competing interest None., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Microtubules (MTs), highly dynamic structures composed of α- and β-tubulin heterodimers, are involved in cell movement and intracellular traffic and are essential for cell division. Within the cell, MTs are not uniform as they can be composed of different tubulin isotypes that are post-translationally modified and interact with different microtubule-associated proteins (MAPs). These diverse intrinsic factors influence the dynamics of MTs. Extrinsic factors such as microtubule-targeting agents (MTAs) can also affect MT dynamics. MTAs can be divided into two main categories: microtubule-stabilizing agents (MSAs) and microtubule-destabilizing agents (MDAs). Thus, the MT skeleton is an important target for anticancer therapy. This review discusses factors that determine the microtubule dynamics in normal and cancer cells and describes microtubule-MTA interactions, highlighting the importance of tubulin isoform diversity and post-translational modifications in MTA responses and the consequences of such a phenomenon, including drug resistance development.

Katanin-like 2 protein (Katnal2) orthologs have a tripartite domain organization. Two highly conserved regions, an N-terminal LisH (Lis-homology) domain and a C-terminal AAA catalytic domain, are separated by a less conserved linker. The AAA domain of Katnal2 shares the highest amino acid sequence homology with the AAA domain of the canonical katanin p60. Katnal2 orthologs are present in a wide range of eukaryotes, from protists to humans. In the ciliate Tetrahymena thermophila , a Katnal2 ortholog, Kat2, co-localizes with the microtubular structures, including basal bodies and ciliary outer doublets, and this co-localization is sensitive to levels of microtubule glutamylation. The functional analysis of Kat2 domains suggests that an N-terminal fragment containing a LisH domain plays a role in the subcellular localization, dimerization, and stability of Kat2., Competing Interests: The authors declare no conflicts of interest.

Primary ciliary dyskinesia (PCD) is a recessive heterogeneous disorder of motile cilia, affecting one per 15,000-30,000 individuals; however, the frequency of this disorder is likely underestimated. Even though more than 40 genes are currently associated with PCD, in the case of approximately 30% of patients, the genetic cause of the manifested PCD symptoms remains unknown. Because motile cilia are highly evolutionarily conserved organelles at both the proteomic and ultrastructural levels, analyses in the unicellular and multicellular model organisms can help not only to identify new proteins essential for cilia motility (and thus identify new putative PCD-causative genes), but also to elucidate the function of the proteins encoded by known PCD-causative genes. Consequently, studies involving model organisms can help us to understand the molecular mechanism(s) behind the phenotypic changes observed in the motile cilia of PCD affected patients. Here, we summarize the current state of the art in the genetics and biology of PCD and emphasize the impact of the studies conducted using model organisms on existing knowledge., Competing Interests: The authors declare no conflicts of interest.

The outer and inner dynein arms (ODAs and IDAs) are composed of multiple subunits including dynein heavy chains possessing a motor domain. These complex structures are preassembled in the cytoplasm before being transported to the cilia. The molecular mechanism(s) controlling dynein arms' preassembly is poorly understood. Recent evidence suggests that canonical R2TP complex, an Hsp-90 co-chaperone, in cooperation with dynein axonemal assembly factors (DNAAFs), plays a crucial role in the preassembly of ODAs and IDAs. Here, we have summarized recent data concerning the identification of novel chaperone complexes and their role in dynein arms' preassembly and their association with primary cilia dyskinesia (PCD), a human genetic disorder.

Cilia are highly evolutionarily conserved, microtubule-based cell protrusions present in eukaryotic organisms from protists to humans, with the exception of fungi and higher plants. Cilia can be broadly divided into non-motile sensory cilia, called primary cilia, and motile cilia, which are locomotory organelles. The skeleton (axoneme) of primary cilia is formed by nine outer doublet microtubules distributed on the cilium circumference. In contrast, the skeleton of motile cilia is more complex: in addition to outer doublets, it is composed of two central microtubules and several diverse multi-protein complexes that are distributed periodically along both types of microtubules. For many years, researchers have endeavored to fully characterize the protein composition of ciliary macro-complexes and the molecular basis of signal transduction between these complexes. Genetic and biochemical analyses have suggested that several hundreds of proteins could be involved in the assembly and function of motile cilia. Within the last several years, the combined efforts of researchers using cryo-electron tomography, genetic and biochemical approaches, and diverse model organisms have significantly advanced our knowledge of the ciliary structure and protein composition. Here, we summarize the recent progress in the identification of the subunits of ciliary complexes, their precise intraciliary localization determined by cryo-electron tomography data, and the role of newly identified proteins in cilia.

Ciliopathies are a group of genetic diseases caused by defects in the function of cilia, that are cellular processes composed of a microtubule-based core. Ciliopathies present with pathological changes in one or many organs at the same time. Symptoms of ciliopathies depend on the type of damaged tissues and organs. The most common are polycystic kidney and liver, blindness, dysfunction of neural tube, brain anomalies, mental retardation, abnormalities in skeletal system from polydactyly to abnormal short ribs and limbs, abnormalities in ectoderms, obesity, situs inversus, infertility and infections of the upper airways. Both basic and clinical studies provide data regarding novel ciliary proteins the lack or mutation of which are associated with cilia dysfunction and which, in consequence, may give rise to ciliopathies. The number of ciliopathies (35 known at present) is still increasing due to identification of additional genes (187 identified up to now) directly connected with these diseases. In this work, the most important mechanisms responsible for abnormal cilia formation and functioning, that constitute the primary cause of ciliopathies, are presented.

Cilia beating is powered by the inner and outer dynein arms (IDAs and ODAs). These multi-subunit macrocomplexes are arranged in two rows on each outer doublet along the entire cilium length, except its distal end. To generate cilia beating, the activity of ODAs and IDAs must be strictly regulated locally by interactions with the dynein arm-associated structures within each ciliary unit and coordinated globally in time and space between doublets and along the axoneme. Here, we provide evidence of a novel ciliary complex composed of two conserved WD-repeat proteins, Fap43p and Fap44p. This complex is adjacent to another WD-repeat protein, Fap57p, and most likely the two-headed inner dynein arm, IDA I1. Loss of either protein results in altered waveform, beat stroke and reduced swimming speed. The ciliary localization of Fap43p and Fap44p is interdependent in the ciliate Tetrahymena thermophila.

The mechanisms that regulate γ-tubulin, including its post-translational modifications, are poorly understood. γ-Tubulin is important for the duplication of centrioles and structurally similar basal bodies (BBs), organelles which contain a ring of nine triplet microtubules. The ciliate Tetrahymena thermophila carries hundreds of cilia in a single cell and provides an excellent model to specifically address the role of γ-tubulin in the BBs assembly and maintenance. The genome of Tetrahymena contains a single γ-tubulin gene. We show here that there are multiple isoforms of γ-tubulin that are likely generated by post-translational modifications. We identified evolutionarily conserved serine and threonine residues as potential phosphosites of γ-tubulin, including S80, S129, S131, T283, and S360. Several mutations that either prevent (S80A, S131A, T283A, S360A) or mimic (T283D) phosphorylation were conditionally lethal and at a higher temperature phenocopied a loss of γ-tubulin. Cells that overproduced S360D γ-tubulin displayed phenotypes consistent with defects in the microtubule-dependent functions, including an asymmetric division of the macronucleus and abnormalities in the pattern of BB rows, including gaps, fragmentation, and misalignment. In contrast, overexpression of S129D γ-tubulin affected the orientation, docking, and structure of the BBs, including a loss of either the B- or C-subfibers or the entire triplets. We conclude that conserved potentially phosphorylated amino acids of γ-tubulin are important for either the assembly or stability of BBs., (© 2018 Wiley Periodicals, Inc.)

Serotonin (5-hydroxytryptamine; 5-HT) plays an important role in control of locomotion, partly through direct effects on motoneurons. Spinal cord complete transection (SCI) results in changes in 5-HT receptors on motoneurons that influence functional recovery. Activation of 5-HT 2A and 5-HT 7 receptors improves locomotor hindlimb movements in paraplegic rats. Here, we analyzed the mRNA of 5-HT 2A and 5-HT 7 receptors (encoded by Htr2a and Htr7 genes, resp.) in motoneurons innervating tibialis anterior (TA) and gastrocnemius lateralis (GM) hindlimb muscles and the tail extensor caudae medialis (ECM) muscle in intact as well as spinal rats. Moreover, the effect of intraspinal grafting of serotonergic neurons on Htr2a and Htr7 gene expression was examined to test the possibility that the graft origin 5-HT innervation in the spinal cord of paraplegic rats could reverse changes in gene expression induced by SCI. Our results indicate that SCI at the thoracic level leads to changes in Htr2a and Htr7 gene expression, whereas transplantation of embryonic serotonergic neurons modifies these changes in motoneurons innervating hindlimb muscles but not those innervating tail muscles. This suggests that the upregulation of genes critical for locomotor recovery, resulting in limb motoneuron plasticity, might account for the improved locomotion in grafted animals.

Microtubules are hollow tube-like polymeric structures composed of α,β-tubulin heterodimers. They play an important role in numerous cellular processes, including intracellular transport, cell motility and segregation of the chromosomes during cell division. Moreover, microtubule doublets or triplets form a scaffold of a cilium, centriole and basal body, respectively. To perform such diverse functions microtubules have to differ in their properties. Post-translational modifications are one of the factors that affect the properties of the tubulin polymer. Here we focus on the direct and indirect effects of post-translational modifications of tubulin on microtubule dynamics., Competing Interests: The authors declare no conflict of interest.

PhLP2 is a small cytosolic protein that belongs to the highly conserved phosducin-like family of proteins. In amniote genomes there are two PhLP2 homologs, PhLP2A and PhLP2B. It has been shown that mammalian PhLP2A modulates the CCT/TRiC chaperonin activity during folding of cytoskeletal proteins. In order to better understand the function of PhLP2A in cellular protein quality control system, in the present study we have searched for its protein targets. Applying immunoprecipitation followed by mass spectrometry analysis we have identified Hsp90 as a partner of PhLP2A. With pull down experiments, we have confirmed this interaction in protein lysate and using purified proteins we have shown that PhLP2A interacts directly with Hsp90. Furthermore, the proximity ligation assay (PLA) performed on mIMCD-3 cells has shown that PhLP2A forms complexes with Hsp90 which are mainly localized in the cytoplasm of these cells. Further analysis has indicated that the level of PhLP2A increases after heat shock or radicicol treatment, similarly as the level of Hsp90, and that expression of PhLP2A after heat shock is regulated at the transcriptional level. Moreover, using recombinant luciferase we have shown that PhLP2A stabilizes this enzyme in a folding competent state and prevents its denaturation and aggregation. In addition, overexpression of PhLP2A in HEK-293 cells leads to increased heat stress resistance. Altogether, our results have shown that PhLP2A interacts with Hsp90 and exhibits molecular chaperone activity toward denatured proteins. J. Cell. Biochem. 118: 420-429, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Katanin is a microtubule severing protein that functions as a heterodimer composed of an AAA domain catalytic subunit, p60, and a regulatory subunit, a WD40 repeat protein, p80. Katanin-dependent severing of microtubules is important for proper execution of key cellular activities including cell division, migration, and differentiation. Published data obtained in Caenorhabditis elegans, Xenopus and mammals indicate that katanin is regulated at multiple levels including transcription, posttranslational modifications (of both katanin and microtubules) and degradation. Little is known about how katanin is regulated in unicellular organisms. Here we show that in the ciliated protist Tetrahymena thermophila, as in Metazoa, the localization and activity of katanin requires specific domains of both p60 and p80, and that the localization of p60, but not p80, is sensitive to the levels of microtubule glutamylation. A prolonged overexpression of either a full length, or a fragment of p80 containing WD40 repeats, partly phenocopies a knockout of p60, indicating that in addition to its activating role, p80 could also contribute to the inhibition of p60. We also show that the level of p80 depends on the 26S proteasome activity., (© 2016 John Wiley & Sons Ltd.)

The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery.

Bacterial mechano-sensitive (MS) channels reside in the inner membrane and are considered to act as emergency valves whose role is to lower cell turgor when bacteria enter hypo-osmotic environments. However, there is emerging evidence that members of the Mechano-sensitive channel Small (MscS) family play additional roles in bacterial and plant cell physiology. MscS has a large cytoplasmic C-terminal region that changes its shape upon activation and inactivation of the channel. Our pull-down and co-sedimentation assays show that this domain interacts with FtsZ, a bacterial tubulin-like protein. We identify point mutations in the MscS C-terminal domain that reduce binding to FtsZ and show that bacteria expressing these mutants are compromised in growth on sublethal concentrations of β-lactam antibiotics. Our results suggest that interaction between MscS and FtsZ could occur upon inactivation and/or opening of the channel and could be important for the bacterial cell response against sustained stress upon stationary phase and in the presence of β-lactam antibiotics.

Dynein motors and regulatory complexes repeat every 96 nm along the length of motile cilia. Each repeat contains three radial spokes, RS1, RS2, and RS3, which transduct signals between the central microtubules and dynein arms. Each radial spoke has a distinct structure, but little is known about the mechanisms of assembly and function of the individual radial spokes. In Chlamydomonas, calmodulin and spoke-associated complex (CSC) is composed of FAP61, FAP91, and FAP251 and has been linked to the base of RS2 and RS3. We show that in Tetrahymena, loss of either FAP61 or FAP251 reduces cell swimming and affects the ciliary waveform and that RS3 is either missing or incomplete, whereas RS1 and RS2 are unaffected. Specifically, FAP251-null cilia lack an arch-like density at the RS3 base, whereas FAP61-null cilia lack an adjacent portion of the RS3 stem region. This suggests that the CSC proteins are crucial for stable and functional assembly of RS3 and that RS3 and the CSC are important for ciliary motility., (© 2015 Urbanska, Song, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

In Tetrahymena, besides apparent cell polarity generated by specialized cortical structures, several proteins display a specific asymmetric distribution suggesting their involvement in the generation and the maintenance of cell polarization. One of these proteins, a membrane skeleton protein called fenestrin, forms an antero-posterior gradient, and is accepted as a marker of cell polarity during different cellular processes, such as cell division or oral replacement. In conjugating cells, fenestrin forms an intracytoplasmic net which participates in pronuclear exchange. The function of fenestrin is still unknown. To better understand the role of fenestrin we characterized this protein in an amicronuclear Tetrahymena pyriformis. We show that in this ciliate not only does fenestrin localization change in a cell division-dependent manner, but its mRNA and protein level is also cell cycle-regulated. We determine that the two available anti-fenestrin antibodies, 3A7 and 9A7, recognize different pools of fenestrin isoforms, and that 9A7 is the more general. In addition, our results indicate that fenestrin is a phosphoprotein. We also show that the level of fenestrin in the amicronuclear T. pyriformis and the amicronuclear BI3840 strain of T. thermophila is several times lower than in micronuclear T. thermophila., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Recent studies have implicated the phosducin-like protein-2 (PHLP2) in regulation of CCT, a chaperonin whose activity is essential for folding of tubulin and actin. However, the exact molecular function of PHLP2 is unclear. Here we investigate the significance of PHLP2 in a ciliated unicellular model, Tetrahymena thermophila, by deleting its single homolog, Phlp2p. Cells lacking Phlp2p became larger and died within 96 h. Overexpressed Phlp2p-HA localized to cilia, basal bodies, and cytosol without an obvious change in the phenotype. Despite similar localization, overexpressed GFP-Phlp2p caused a dominant-negative effect. Cells overproducing GFP-Phlp2p had decreased rates of proliferation, motility and phagocytosis, as compared to wild type cells or cells overproducing a non-tagged Phlp2p. Growing GFP-Phlp2p-overexpressing cells had fewer cilia and, when deciliated, failed to regenerate cilia, indicating defects in cilia assembly. Paclitaxel-treated GFP-Phlp2p cells failed to elongate cilia, indicating a change in the microtubules dynamics. The pattern of ciliary and cytosolic tubulin isoforms on 2D gels differed between wild type and GFP-Phlp2p-overexpressing cells. Thus, in Tetrahymena, PhLP2 is essential and under specific experimental conditions its activity affects tubulin and microtubule-dependent functions including cilia assembly., (Copyright © 2013 Wiley Periodicals, Inc.)

The calcyclin binding protein and Siah-1 interacting protein (CacyBP/SIP) protein was shown to play a role in the organization of microtubules. In this work we have examined the neuronal distribution and possible function of CacyBP/SIP in cytoskeletal pathophysiology. We have used brain tissue from Alzheimer's disease (AD) patients and from transgenic mice modeling 2 different pathologies characteristic for AD: amyloid and tau. In the brain from AD patients, CacyBP/SIP was found to be almost exclusively present in neuronal somata, and in control patients it was seen in the somata and neuronal processes. In mice doubly transgenic for amyloid precursor protein and presenilin 1 there was no difference in CacyBP/SIP neuronal localization in comparison with the nontransgenic animals. By contrast in tau transgenic mice, localization of CacyBP/SIP was similar to that observed for AD patients. To find the relation between CacyBP/SIP and tau we examined dephosphorylation of tau by CacyBP/SIP. We found that indeed it exhibited phosphatase activity toward tau. Altogether, our results suggest that CacyBP/SIP might play a role in AD pathology., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The coloured ciliate Blepharisma japonicum changes swimming velocity (positive photokinesis) and elongates its body in response to a prolonged illumination. We have recently proposed that alterations in the phosphorylation level of the ciliate phosducin (Pdc) may be involved in light-induced cell elongation, which in turn affects the interaction of βγ-dimer of G-proteins (Gβγ) with β-tubulin and subsequent cytoskeletal remodelling. The cellular mechanism that governs the photokinetic effect in this ciliate has not been elucidated. In the present study, we utilise real-time PCR to demonstrate that the levels of ciliate Pdc mRNA are significantly reduced in Pdc-RNAi-treated cells compared to cells fed with bacteria carrying the empty vector (control cells). Using western immunoblotting, we confirmed that these cells treated with Pdc-RNAi expressed a substantially lower level of the Pdc protein. The assay also revealed that in ciliates treated with Pdc-RNAi and exposed to light, the cytosolic level of Gβ (~36 kDa) was reduced, whereas the level of Gβ localized to the membrane (~32 kDa) was increased compared to control cells. In addition, behavioural analysis of the cells indicated a substantial reduction of photokinesis. The findings in this study provide additional characterization of the functional properties of the ciliate Pdc protein and we discuss a likely role for this phosphoprotein in the photokinetic phenomenon of the ciliate protist Blepharisma.

Blepharisma japonicum ciliates display reversible cell elongation in response to lasting bright illumination. This light-induced phenomenon has been ascribed to the active sliding of the cortical microtubules of the ciliate. The detailed intracellular signaling pathway that activates the microtubule network in response to light, resulting in cell elongation, is unknown. We have previously reported that light stimulation initiates sequential molecular events consisting of a decrease in the phosphorylation of ciliate Pdc, followed by increased binding of Pdc to membrane-localised Gbetagamma and the subsequent translocation of the Pdc-Gbetagamma complex to the cytoplasm. In this study, we used selected agents known to influence protein phosphorylation to test whether alterations in Pdc phosphorylation levels by light affect ciliate shape. Behavioural analysis indicated that cell treatment with okadaic acid, an inhibitor of protein phosphatase activity, heavily abolished the effect of light on cell elongation, whereas the presence of H-89, a specific inhibitor of cAMP-dependent protein kinase (PKA) activity, had no appreciable effect on the cell length. Phosphorylation assays showed that cell incubation with H-89 mimicked light by promoting Pdc dephosphorylation and its colocalization with Gbetagamma. However, as demonstrated by FRET-AP, Pdc-Gbetagamma complex formation and changes in the length of the cell did not occur under the same conditions. Moreover, fluorescence microscopy showed localization of Gbetagamma and beta-tubulin in the same cell compartment and demonstrated that a direct interaction between these proteins occurs in cells adapted to darkness or exposed to prolonged illumination (> or = 10 min). In contrast, an opposite effect, i.e. a transient decrease in the interaction between Gbetagamma and beta-tubulin and distinct Pdc dephosphorylation, was observed in cells illuminated for short time. Under these conditions, Pdc preferentially occupies the cell submembrane region and interacts with Gbetagamma. In cells illuminated for a longer time (> or = 10 min) and despite the constant light intensity, Pdc was progressively rephosphorylated and then dissociated from Gbetagamma, relocalizing within the cell cytoplasm. The results obtained in this study suggest that alterations in Pdc phosphorylation may be involved in light-induced elongation of the Blepharisma cell body, which affects the interaction of Gbetagamma with beta-tubulin and cell cytoskeleton remodelling.

Immunoblotting of isolated cell membrane fractions from ciliates Blepharisma japonicum and Stentor coeruleus with a polyclonal antibody raised against rhodopsin revealed one strong protein band of about 36 kDa, thought to correspond to protozoan rhodopsin. Inspection of both ciliates labeled with the same antibody using a confocal microscope confirmed the immunoblotting result and demonstrated the presence of these rhodopsin-like molecules localized within the cell membrane area. Immunoblot analysis of the ciliate membrane fractions resolved by two-dimensional gel electrophoresis identified two distinct 36 kDa spots at pIs of 4.5 and 7.0 for Blepharisma, and three spots at pIs of 4.4, 5.0 and 6.0 for Stentor, indicating a possible mixture of phosphorylated rhodopsin species in these cells. The obtained results suggest that both Blepharisma and the related ciliate Stentor contain within the cell membrane the rhodopsin-like proteins, which may be involved as receptor molecules in the sensory transduction pathway mediating motile photoresponses in these protists as in other species of lower eukaryota.

Chaperonins are large oligomers consisting of two superimposed rings, each enclosing a cavity used for the folding of other proteins. They have been divided into two groups. Chaperonins of type I were identified in mitochondria and chloroplasts (Hsp60) or bacterial cytosol (GroEL) as well. Chaperonins type II were found in Archea and the eukaryotic cell cytosol (CCT). Protein folding occurs in the chaperonin after its conformational changes induced upon ATP binding. Mechanism of the protein folding, although still poorly defined, clearly differs from the one established for GroEL. Although CCT with prefoldin seems to be mainly involved in the folding of actin and tubulin, other substrates engaged in various cellular processes are beginning to be characterized, including proteins possessing WD40-repeats. Moreover, several lines of evidence suggest that beside prefoldin, CCT may work in concert with phosducin-like proteins (PhLPs).

The aim of this study was to search for a mechanism responsible for the acquisition of cell polarity in a ciliate Tetrahymena. Homologs of the mammalian genes coding for CDC42-GSK3beta- MARK/PAR1-MAPs proteins were found in the Tetrahymena genome (Eisen et al., 2006, and this study). These proteins belong to a pathway which controls assembly and disassembly of microtubule bundles and cell polarity in neural cells. In Tetrahymena, there are two types of morphogenesis: divisional and oral replacement (OR). In divisional morphogenesis, an elongation of longitudinal microtubule bundles (LMs) takes place during cell division. In contrast, in OR type morphogenesis, which occurs in starved non-dividing cells, a polar retraction of LMs occurs. In T. pyriformis, the frequency of developmental switch to OR morphogenesis increases in the presence of wortmannin, an inhibitor of the CDC42-GSK3beta-MARK pathway. In contrast, wortmannin when applied to dividing cells does not affect divisional morphogenesis. Using immunostaining with the antibody against mammalian mitotic phosphoproteins (MPM-2) we show that these proteins co-localize with the LMs and are distributed along the anterior-posterior gradient. In addition, we show that during OR type morphogenesis, the fate of LMs correlates with the anterior-posterior gradient of instability of the cortical structures. We used the conditional mouth-less mutant of T. thermophila (Tiedtke et al., 1988) to test if the presence of the oral apparatus is required for the maintenance of cell polarity. We discuss our results in relation to the hypothesis of GSK3-beta-MARK pathway involvement in the acquisition of cell polarity in Tetrahymena.

Immunological techniques and high-resolution FRET analysis were employed to investigate the in vivo colocalization and interaction of phosducin (Pdc) with the betagamma-subunits of G-protein (Gbetagamma) in the ciliate Blepharisma japonicum. Immunological techniques revealed that illumination of cells resulted in a decrease in phosphorylation levels of Pdc and its colocalization with Gbetagamma. The observed light-induced Pdc dephosphorylation was also accompanied by significant enhancement of Gbetagamma binding by this molecule. Possible formation of the Pdc-Gbetagamma complex in cells exposed to light was corroborated by FRET between these proteins. Treatment of cells with okadaic acid, an inhibitor of phosphatase activity, entirely prevented Pdc dephosphorylation by light, colocalization of this phosphoprotein with Gbetagamma and generation of the Pdc-Gbetagamma complex. Cell fractionation and immunoblotting revealed that in cells exposed to light, the formation of Pdc-Gbetagamma complex and its translocation into the cytoplasm occur simultaneously with a change in the gel migration of Gbeta. Moreover, a 33 kDa immunoanalog of 14-3-3 protein was identified and we showed that this protein is bound by phosphorylated Pdc in a cell adapted to darkness. The results of this study provide additional detailed characterization of the functional properties of the ciliate Pdc. The likely functional role of Pdc in Blepharisma is discussed.

Blepharisma japonicum and Stentor coeruleus are related ciliates, conspicuous by their photosensitivity. They are capable of avoiding illuminated areas in the surrounding medium, gathering exclusively in most shaded places (photodispersal). Such behaviour results mainly from motile photophobic response occurring in ciliates. This light-avoiding response is observed during a relatively rapid increase in illumination intensity (light stimulus) and consists of cessation of cell movement, a period of backward movement (ciliary reversal), followed by a forward swimming, usually in a new direction. The photosensitivity of ciliates is ascribed to their photoreceptor system, composed of pigment granules, containing the endogenous photoreceptor -- blepharismin in Blepharisma japonicum, and stentorin in Stentor coeruleus. A light stimulus, applied to both ciliates activates specific stimulus transduction processes leading to the electrical changes at the plasma membrane, correlated with a ciliary reversal during photophobic response. These data indicate that both ciliates Blepharisma japonicum and Stentor coeruleus, the lower eukaryotes, are capable of transducing the perceived light stimuli in a manner taking place in some photoreceptor cells of higher eukaryotes. Similarities and differences concerning particular stages of light transduction in eukaryotes at different evolutional levels are discussed in this article.

We have previously reported that motile photophobic response in ciliate Blepharisma japonicum correlates with dephosphorylation of a cytosolic 28 kDa phosphoprotein (PP28) exhibiting properties similar to those of phosducin. Here we demonstrate in in vivo phosphorylation assay that the light-elicited dephosphorylation of the PP28 is significantly modified by cell incubation with substances known to modulate protein phosphatase and kinase activities. Immunoblot analyses showed that incubation of ciliates with okadaic acid and calyculin A, potent inhibitors of type 1 or 2A protein phosphatases, distinctly increased phosphorylation of PP28 in dark-adapted cells and markedly weakened dephosphorylation of the ciliate phosducin following cell illumination. An enhancement of PP28 phosphorylation was also observed in dark-adapted ciliates exposed to 8-Br-cAMP and 8-Br-cGMP, slowly hydrolysable cyclic nucleotide analogs and 3-isobutyryl-1-methylxanthine (IBMX), a non-specific cyclic nucleotide phosphodiesterase (PDEs) inhibitor. Only slight changes in light-evoked dephosphorylation levels of PP28 were observed in cells treated with the cyclic nucleotide analogs and IBMX. Incubation of ciliates with H 89 or KT 5823, highly selective inhibitor of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), respectively, decreased PP28 phosphorylation levels in dark-adapted cells, whereas the extent of light-evoked dephosphorylation of the phosphoprotein was only slightly influenced. Cell treatment with higher Ca2+ concentration together with ionophore A23187 in culture medium resulted in marked increase in PP28 phosphorylation levels, while quite an opposite effect was observed in cells exposed to Ca2+ chelators, EGTA or BAPTA/AM as well as calmodulin antagonists, such as trifluoperazine (TFP), W-7 or calmidazolium. Light-dependent dephosphorylation was not considerably affected by these treatments. The experimental findings presented here suggest that an endogenous light-dependent protein kinase-phosphatase system may be engaged in the alteration of phosducin phosphorylation in ciliate B. japonicum thereby to modulate the cell motile photophobic behavior.

G protein-coupled receptor kinases (GRKs) are key modulators of G protein-coupled receptor (GPCR) signaling. They constitute a family of seven mammalian serine-threonine protein kinases that phosphorylate agonist-bound receptor. GRKs-mediated receptor phosphorylation rapidly initiates profound impairment of receptor signaling and desensitization. Activity of GRKs and subcellular targeting is tightly regulated by interaction with receptor domains, G protein subunits, lipids, anchoring proteins and calcium sensitive proteins. Moreover, GRK phosphorylation by several other kinases and autophosphorylation have recently been shown to modulate its functionality. This review summarize our current knowledge of GRKs regulatory mechanisms and GRKs physiological function.

Examination of ciliate Blepharisma japonicum whole cell lysates with an antibody against phosphoserine and in vivo labeling of cells with radioactive phosphate revealed that the photophobic response in the ciliate is accompanied by a rapid dephosphorylation of a 28 kDa protein and an enhanced phosphorylation of a 46 kDa protein. Analysis with antibodies raised against rat phosducin or human phosducin-like proteins, identified one major protein of a molecular weight of 28 kDa, and two protein bands of 40 kDa and 93 kDa. While the identified ciliate phosducin is phosphorylated in a light-dependent manner, both phosducin-like proteins exhibit no detectable dependence of phosphorylation upon illumination. An immunoprecipitation assay also showed that the ciliate phosducin is indeed phosphorylated on a serine residue and exists in a phosphorylated form in darkness and that its dephosphorylation occurs in light. Immunocytochemical experiments showed that protozoan phosducin and phosducin-like proteins are localized almost uniformly within the cytoplasm of cells adapted to darkness. Cell exposure to light caused a pronounced displacement of the cell phosducin to the vicinity of the plasma membrane; however, no translocation of phosducin-like proteins was observed upon cell illumination. The obtained results are the first demonstration of the presence and morphological localization of a possible phosducin and phosducin-like proteins in ciliate protists. Phosducin and phosducin-like proteins were found to bind and sequester the betagamma-subunits of G-proteins with implications for regulation of G-protein-mediated signaling pathways in various eukaryotic cells. The findings presented in this study suggest that the identified phosphoproteins in photosensitive Blepharisma japonicum may also participate in the regulation of the efficiency of sensory transduction, resulting in the motile photophobic response in this cell.

The protozoan ciliate Stentor coeruleus displays a step-up photophobic response to an increase in light intensity in its environment. The motile response consists of a delayed stop of ciliary beating and transient ciliary reversal period. Such light-avoiding behavior was significantly influenced by an incubation of cells with l-cis-diltiazem, a common blocker of cyclic guanosine monophosphate (cGMP)-gated ion channel conductance. The introduction of l-cis-diltiazem to the medium induced ciliary reversal in control cells, mimicking the step-up photophobic response. In light-stimulated ciliates, the presence of this inhibitor caused a substantial decrease of the latency of ciliary stop response, prolongation of the ciliary reversal duration and also an increase of cell photoresponsiveness in a dose- and time-dependent manner. The obtained behavioral results support the suggestion that the photosensitive ciliate S. coeruleus possesses cGMP-gated channels, which may be involved in the process of light signal transduction for the motile photophobic response.

It is widely known that the phototransduction process in vertebrate photoreceptor cells are mediated by cGMP-gated ionic channels. The recent electrophysiological study showed that the cGMP-gated channels are also present in light sensitive protozoan ciliate Stentor coeruleus. These channels might play a key role in phototransduction process which leads to the photophobic behavior in the ciliate. The basic biophysical and pharmaceutical properties of cGMP-gated channels in Stentor indicate high similarity to those in vertebrate photoreceptor cells.

Abstract- Photoinduced formation of inositol 1,4,5-trisphosphate (Ins[1,4,5]P 3 ) was examined using a specific radioimmu-noassay to investigate the molecular mechanisms of light signal transduction mediating photophobic responses in the ciliate Blepharisma japonicum. Application of light stimuli of moderate intensity to dark-adapted cells induced a rapid and significant increase in the basal level of Ins (1,4,5)P 3 , with a peak at about 20 s. Thereafter, the level of Ins (1,4,5)P 3 declined to the resting value within the subsequent 100 s. Light stimuli of higher intensity raised the cell Ins (1,4,5)P 3 content to still higher levels within about 20 s, but the decaying time course was considerably prolonged. In ciliates incubated under dark conditions with agents interfering with the inositol signalling pathway, like neomycin and Li + the basal levels of Ins (1,4,5)P 3 were lower than in control cells. A photoinduced rise of Ins (1,4,5)P 3 , content in ciliates treated with neomycin or Li + was significantly inhibited in a dose-dependent manner. Depolarizing ionic stimuli in dark-adapted ciliates induced no significant alterations of the resting Ins (1,4,5)P 3 level, indicating a lack of a contribution of this kind of stimulation to the inositol turnover. These studies are the first in vivo demonstration of a possible role for inositol trisphosphate as a second messenger in the light signal transduction process in the ciliate B. japonicum.

The effect of various modulators of cytoplasmic guanosine 3',5'-cyclic monophosphate (cGMP) level on the step-up photophobic responses in Blepharisma japonicum has been investigated to clarify the possible role of cGMP in the mechanism of photosensory signal transduction. Membrane-permeable analogs of cGMP, 8-bromo-guanosine 3',5'-cyclic monophosphate or dibutyryl cGMP, caused a marked dose-dependent prolongation of the latency for the photophobic response, resulting in inhibition of the photophobic response in Blepharisma japonicum. A similar effect was observed when cells were treated with 3'-isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, and pertussis toxin, a G-protein activity modulator. The G-protein activator, fluoroaluminate, and 6-anilino-5,8-quinolinedione (LY 83583), an agent which effectively lowers the cytoplasmic cGMP level, significantly enhanced the photoresponsiveness of these ciliates to visible light stimuli. These results suggest that cellular cGMP serves as a signal modulator in the photophobic response of Blepharisma japonicum.

Light-induced membrane potential changes and motile responses have been studied in Stentor cells with intracellular microelectrodes and video microscopy, respectively. Intracellular microelectrode recordings showed that step-up increase in light intensity induced an electrical membrane response which consisted of an initial membrane depolarization (photoreceptor potential) followed by an action potential and maintaining phase of depolarization (afterdepolarization). The amplitude of the receptor potential is dependent on the intensity of light stimulus and the action potential appears with a lag period (latency) after the onset of light stimulus. The extent of the membrane afterdepolarization is dependent on the intensity and duration of stimulus used. A close time correlation has been established between the latency for the action potential and the onset of ciliary reversal (stop response). A time correlation was also observed between the duration of the membrane afterdepolarization and the duration of backward swimming. The action spectrum for the photoreceptor potential amplitude of Stentor resembled the action spectra for the latency of ciliary reversal and the photoresponsiveness, indicating that the photomovement response and membrane potential changes are coupled through the same photosensor system. A hypothesis on the photosensory transduction chain in Stentor is discussed according to which the photoreceptors and the ciliary apparatus is mediated by the membrane potential changes.

The heterotrichous ciliate, Stentor coeruleus, exhibits a well-defined photophobic response to a sudden increase in the intensity of visible light. The phobic reactions usually appear with a latency period (i.e. a time delay between the onset of the stimulus and the stop response). This latency of phobic response was significantly increased when the cells were incubated with 8-bromo-guanosine 3',5'-cyclic monophosphate. In the presence of this nucleotide, a reduction of cell responsiveness (i.e. the number of photophobically responding cells) was also observed. Similar effects were observed when cells were treated with pertussis toxin, a G-protein activity modulator, and 3'-isobutyl-methylxanthine, an inhibitor of guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase. The G-protein activator fluoroaluminate and 6-anilino-5,8-quinolinedione (LY 83583) (an effective agent for lowering cellular cGMP levels) showed opposite effects on the cell photophobic response. These results indirectly suggest that the level of cytoplasmic cGMP, possibly modulated by a G-protein-coupled cGMP phosphodiesterase, plays a phototransducing role in Stentor. In addition, using an antiserum raised against bovine transducin, a cross-reacting protein with an apparent molecular mass of 39 kDa was detected on immunoblots. The alpha-subunit of a Stentor G-protein has also been partially cloned and sequenced. However, the possible coupling between the G-protein and the putative phosphodiesterase remains to be established.