First-in-Class Colchicine-Based Visible Light Photoswitchable Microtubule Dynamics Disrupting Agent.

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.

Systematic Studies on Anti-Cancer Evaluation of Stilbene and Dibenzo[b,f]oxepine Derivatives.

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 ß-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.

PCD Genes-From Patients to Model Organisms and Back to Humans.

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.

Composition and function of the C1b/C1f region in the ciliary central apparatus.

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.

Intrinsic and Extrinsic Factors Affecting Microtubule Dynamics in Normal and Cancer Cells.

Microtubules (MTs), highly dynamic structures composed of α- and ß-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.

CacyBP/SIP in the rat spinal cord in norm and after transection - Influence on the phosphorylation state of ERK1/2 and p38 kinases.

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.

The LisH Domain-Containing N-Terminal Fragment is Important for the Localization, Dimerization, and Stability of Katnal2 in Tetrahymena.

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.

Role of the Novel Hsp90 Co-Chaperones in Dynein Arms' Preassembly.

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.

Rare Human Diseases: Model Organisms in Deciphering the Molecular Basis of Primary Ciliary Dyskinesia.

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.

Ciliary Proteins: Filling the Gaps. Recent Advances in Deciphering the Protein Composition of Motile Ciliary Complexes.

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.

Intraspinal Grafting of Serotonergic Neurons Modifies Expression of Genes Important for Functional Recovery in Paraplegic Rats.

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-HT2A and 5-HT7 receptors improves locomotor hindlimb movements in paraplegic rats. Here, we analyzed the mRNA of 5-HT2A and 5-HT7 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.

Multiple phosphorylation sites on γ-tubulin are essential and contribute to the biogenesis of basal bodies in Tetrahymena.

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.

Ciliary proteins Fap43 and Fap44 interact with each other and are essential for proper cilia and flagella beating.

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.

[Ciliopaties - diseases caused by abnormal cilia functioning]. / Ciliopatie ­ choroby spowodowane nieprawidlowym funkcjonowaniem rzesek.

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.

Tubulin Post-Translational Modifications and Microtubule Dynamics.

Microtubules are hollow tube-like polymeric structures composed of α,ß-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.

Regulation of katanin activity in the ciliate Tetrahymena thermophila.

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.

Interaction of a Novel Chaperone PhLP2A With the Heat Shock Protein Hsp90.

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.

Calcyclin Binding Protein/Siah-1 Interacting Protein Is a Hsp90 Binding Chaperone.

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.

Cytoplasmic Domain of MscS Interacts with Cell Division Protein FtsZ: A Possible Non-Channel Function of the Mechanosensitive Channel in Escherichia Coli.

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 ß-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 ß-lactam antibiotics.

The CSC proteins FAP61 and FAP251 build the basal substructures of radial spoke 3 in cilia.

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.