The paraflagellar rod of kinetoplastid parasites: From structure to components and function

The role of the eukaryotic flagellum in cell motility is well established but its importance in many other aspects of cell biology, from cell signalling to developmental regulation, is becoming increasingly apparent. In addition to this diversity of function the core structure of the flagellum, which has been inherited from the earliest ancestor of all eukaryotes, is embellished with a range of extra-axonemal structures in many organisms. One of the best studied of these structures is the paraflagellar rod of kinetoplastid protozoa in which the morphological characteristics have been well defined and some of the major protein constituents have been identified. Here we discuss recent advances in the identification of further molecular components of the paraflagellar rod, how these impact on our understanding of its function and regulation and the implications for therapeutic intervention in a number of devastating human pathologies.     

Two-headed outer- and inner-arm dyneins of Leishmania sp bear conserved IQ-like motifs

Dyneins are high molecular weight microtubule based motor proteins responsible for beating of the flagellum. The flagellum is important for the viability of trypanosomes like Leishmania. However, very little is known about dynein and its role in flagellar motility in such trypanosomatid species. Here, we have identified genes in five species of Leishmania that code for outer-arm dynein (OAD) heavy chains α and β, and inner-arm dynein (IAD) heavy chains 1α and 1β using BLAST and MSA. Our sequence analysis indicates that unlike the three-headed outer-arm dyneins of Chlamydomonas and Tetrahymena, the outer-arm dyneins of the genus Leishmania are two-headed, lacking the γ chain like that of metazoans. N-terminal sequence analysis revealed a conserved IQ-like calmodulin binding motif in the outer-arm α and inner-arm 1α dynein heavy chain in the five species of Leishmania similar to Chlamydomonas reinhardtii outer-arm γ. It was predicted that both motifs were incapable of binding calmodulin. Phosphorylation site prediction revealed conserved serine and threonine residues in outer-arm dynein α and inner-arm 1α as putative phosphorylation sites exclusive to Leishmania but not in Trypanosoma brucei suggesting that regulation of dynein activity might be via phosphorylation of these IQ-like motifs in Leishmania sp.     

A one-step method to identify MAP kinase residues involved in inactivation by tyrosine- and dual-specificity protein phosphatases

MAP kinases (MAPKs) are enzymes directly involved in the control of cellular homeostasis in response to external cues, from differentiation and developmental processes to cell transformation. The activation status of MAPKs, both in magnitude and in duration, reflects the balance of phosphorylation at their Thr and Tyr regulatory residues by specific MAPK kinases and their dephosphorylation by inactivating protein serine/threonine phosphatases (PPs) and protein tyrosine phosphatases (PTPs). The dephosphorylation of MAPKs by PTPs relies on molecular docking between the two enzymes at specific interaction sites. Here we outline a one-step method to identify ERK1/2 and p38α mutations that prevent binding and inactivation by PTPs (tyrosine- or dual-specificity phosphatases) based on the use of anti-pTyr antibodies and cell lysis buffers lacking or containing the broad PTP inhibitor sodium orthovanadate (Na₃VO₄).     

Bacterial-like PPP protein phosphatases: Novel sequence alterations in pathogenic eukaryotes and peculiar features of bacterial sequence similarity

Reversible phosphorylation is a widespread modification affecting the great majority of eukaryotic cellular proteins, and whose effects influence nearly every cellular function. Protein phosphatases are increasingly recognized as exquisitely regulated contributors to these changes. The PPP (phosphoprotein phosphatase) family comprises enzymes, which catalyze dephosphorylation at serine and threonine residues. Nearly a decade ago, “bacterial-like” enzymes were recognized with similarity to proteins from various bacterial sources: SLPs (Shewanella-like phosphatases), RLPHs (Rhizobiales-like phosphatases), and ALPHs (ApaH-like phosphatases). A recent article from our laboratory appearing in Plant Physiology characterizes their extensive organismal distribution, abundance in plant species, predicted subcellular localization, motif organization, and sequence evolution. One salient observation is the distinct evolutionary trajectory followed by SLP genes and proteins in photosynthetic eukaryotes vs. animal and plant pathogens derived from photosynthetic ancestors. We present here a closer look at sequence data that emphasizes the distinctiveness of pathogen SLP proteins and that suggests that they might represent novel drug targets. A second observation in our original report was the high degree of similarity between the bacterial-like PPPs of eukaryotes and closely related proteins of the “eukaryotic-like” phyla Myxococcales and Planctomycetes. We here reflect on the possible implications of these observations and their importance for future research.     

Regulation of outer-arm-dynein activity by phosphorylation and control of ciliary beat frequency

Summary Ciliary beating is empowered by a mechanochemical enzyme, dynein, which appears as two rows of projections on doublet microtubules. While inner-arm dyneins modulate beat form, outer-arm dynein empowers ciliary beat and sets beat frequency. Beat frequency is controlled via phosphorylation of outer-arm dynein. UsingParamecium tetraurelia as model system, we have previously identified a regulatory light chain of outer-arm dynein (22S dynein), M_r29 (p29), whose phosphorylation is cAMP-dependent. The phosphorylation state of the p29 in 22 S dynein determines in vitro microtubule translocation velocity. Although in vitro phosphorylation of p29 takes place in a short time, the percent change ist significantly less than the percent change in dynein activation, or in ciliary beat frequency. A potential mechanism that explains how a few activated dyneins can change ciliary beating is discussed.     

Primary ciliary dyskinesia relative protein ZMYND10 is involved in regulating ciliary function and intraflagellar transport in Paramecium tetraurelia

Cilia are highly conserved in most eukaryotes and are regarded as an important organelle for motility and sensation in various species. Cilia are microscopic, hair-like cytoskeletal structures that protrude from the cell surface. The major focus in studies of cilia has been concentrated on the ciliary dysfunction in vertebrates that causes multisymptomatic diseases, which together are referred to as ciliopathies. To date, the understanding of ciliopathies has largely depended on the study of ciliary structure and function in different animal models. Zinc finger MYND-type containing 10 (ZMYND10) is a ciliary protein that was recently found to be mutated in patients with primary ciliary dyskinesia (PCD). In Paramecium tetraurelia, we identified two ZMYND10 genes, arising from a whole-genome duplication. Using RNAi, we found that the depletion of ZMYND10 in P. tetraurelia causes severe ciliary defects, thus provoking swimming dysfunction and lethality. Moreover, we found that the absence of ZMYND10 caused the abnormal localization of the intraflagellar transport (IFT) protein IFT43 along cilia. These results suggest that ZMYND10 is involved in the regulation of ciliary function and IFT, which may contribute to the study of PCD pathogenesis.     

Speculations on the evolution of 9+2 organelles and the role of central pair microtubules

Motility generated by 9+2 organelles, variably called cilia or flagella, evolved before divergence from the last common ancestor of extant eukaryotes. In order to understand better how motility in these organelles is regulated, evolutionary steps that led to the present 9+2 morphology are considered. In addition, recent advances in our knowledge of flagellar assembly, together with heightened appreciation of the widespread role of cilia in sensory processes, suggest that these organelles may have served multiple roles in early eukaryotic cells. In addition to their function as undulating motility organelles, we speculate that protocilia were the primary determinants of cell polarity and directed motility in early eukaryotes, and that they provided the first defined membrane domain for localization of receptors that allowed cells to respond tactically to environmental cues. Initially, motility associated with these protocilia may have been gliding motility rather than the more complex bend propagation. Once these protocilia became functional motile organelles for beating, we believe that addition of an asymmetric central apparatus, capable of transducing signals to dynein motors and altering beat parameters, provided refined directional control in response to tactic signals. This paper presents hypothesized steps in this evolutionary process, and examples to support these hypotheses.     

Protein kinases and protein phosphatases in prokaryotes: a genomic perspective

For many years, the regulation of protein structure and function by phosphorylation and dephosphorylation was considered a relatively recent invention that arose independently in each phylogenetic domain. Over time, however, incidents of apparent domain trespass involving the presence of 'eukaryotic' protein kinases or protein phosphatases in prokaryotic organisms were reported with increasing frequency. Today, genomics has provided the means to examine the phylogenetic distribution of 'eukaryotic' protein kinases and protein phosphatases in a comprehensive and systematic manner. The results of these genome searches challenge previous conceptions concerning the origins and evolution of this versatile regulatory mechanism.     

Regulation of cyclin-dependent kinases in Arabidopsis thaliana

In plants, different families of cyclin-dependent kinases (CDKs) and cyclins have been identified, indicating that also in plants the progression through the cell cycle is regulated by CDKs. In all eukaryotes, CDKs exert their activity through well-controlled phosphorylations of specific substrates on serine/threonine residues. Such post-translational modifications are universal mechanisms in signal transduction pathways. They allow the organism to differentiate, regulate growth and/or adapt to environmental changes, the latter being crucial for plants because of their sedentary life-style. This adaptation might explain the occurrence of a special CDK type with plant-specific features. This review focuses on the involvement of plant CDKs in different phases of the cell cycle in Arabidopsis thaliana and outlines their regulation by binding to other proteins, and by phosphorylation and dephosphorylation.     

Regulation of alpha(1)-adrenoceptor-linked phosphoinositide metabolism in cultured glia: involvement of protein phosphatases and kinases

Noradrenaline-stimulated phosphoinositide breakdown in cultured glia was found to be mediated by alpha(1A)-adrenoceptors. The alpha(1A)-selective agonist A61603 was as effective as noradrenaline in eliciting 3H-inositol phosphate (IP) accumulation but was approximately 50-fold more potent. In addition, the use of selective antagonists revealed a clear rank order of potency in the ability of these drugs to reverse the effect of noradrenaline on phosphoinositide breakdown: RS17053 (alpha(1A)-selective) >AH11110A (alpha(1B)-selective)>BMY7378 (alpha(1D)-selective). Pre-treatment of cultured glia with the protein phosphatase inhibitor okadaic acid resulted in a concentration- and time-dependent reduction in noradrenaline-evoked 3H-IP accumulation. This effect was mimicked by, but was not additive with, a phorbol ester, was reversed by protein kinase C (PKC) inhibitors and was not evident in cells which had been PKC depleted. The ability of cell extracts to dephosphorylate radiolabelled glycogen phosphorylase revealed the presence of the phosphatases PP1 and PP2A in almost equal abundance. Okadaic acid pre-treatment of intact cultures elicited a marked reduction in total phosphatase activity, particularly that mediated by PP2A. We also determined the effect of okadaic acid pre-treatment on PKC and cyclic AMP-dependent protein kinase (PKA) activities in these cells. PKC and PKA activities in cell extracts were assessed by determining the incorporation of 32P into histone and kemptide, respectively. Okadaic acid elicited increases in both Ca(2+)-dependent and Ca(2+)-independent PKC activity; in addition, increases in both initial and total PKA activities were also recorded. The effect of okadaic acid on noradrenaline-stimulated 3H-IP accumulation were not, however, mimicked by either forskolin or 8-bromo-cyclic AMP, suggesting that this event is not regulated by PKA. Our data point to roles for both PKC and PP2A in the regulation of alpha(1A)-adrenoceptor-linked phosphoinositide metabolism in cultured cortical glia.     

Tubulin evolution: Ciliate-specific epitopes are conserved in the ciliary tubulin of metazoa

Summary In spite of their overall evolutionary conservation, the tubulins of ciliates display electrophoretic and structural particularities. We show here that antibodies raised againstParamecium andTetrahymena ciliary tubulins fail to recognize the cytoplasmic tubulins of all the metazoans tested. Immunoblotting of peptide maps of ciliate tubulins reveals that these antibodies react with one or very few ciliate-specific epitopes, in contrast to polyclonal antibodies against vertebrate tubulins, which are equivalent to autoantibodies and recognize several epitopes in both ciliate and vertebrate tubulins. Furthermore, we show that the anti-ciliate antibodies recognize ciliary and flagellar tubulins of metazoans ranging from sea urchin to mammals (with the exception of humans). The results support the conclusion that although duplication and specialization of tubulin genes in metazoans may have led to distinct types of tubulins, the axonemal one has remained highly conserved.     

Myotubularin phosphoinositide phosphatases, protein phosphatases, and kinases: their roles in junction dynamics and spermatogenesis

Spermatogenesis in the seminiferous epithelium of the mammalian testis is a dynamic cellular event. It involves extensive restructuring at the Sertoli-germ cell interface, permitting germ cells to traverse the epithelium from basal to adluminal compartment. As such, Sertoli-germ cell actin-based adherens junctions (AJ), such as ectoplasmic specializations (ES), must disassemble and reassemble to facilitate this event. Recent studies have shown that AJ dynamics are regulated by intricate interactions between AJ integral membrane proteins (e.g., cadherins, alpha6beta1 integrins and nectins), phosphatases, kinases, adaptors, and the underlying cytoskeleton network. For instance, the myotubularin (MTM) phosphoinositide (PI) phosphatases, such as MTM related protein 2 (MTMR2), can form a functional complex with c-Src (a non-receptor protein tyrosine kinase). In turn, this phosphatase/kinase complex associates with beta-catenin, a constituent of the N-cadherin/beta-catenin functional unit at the AJ site. This MTMR2-c-Src-beta-catenin complex apparently regulates the phosphorylation status of beta-catenin, which determines cell adhesive function conferred by the cadherin-catenin protein complex in the seminiferous epithelium. In this review, we discuss the current status of research on selected phosphatases and kinases, and how these proteins potentially interact with adaptors at AJ in the seminiferous epithelium to regulate cell adhesion in the testis. Specific research areas that are open for further investigation are also highlighted.     

Regulation of ciliary beat frequency by the nitric oxide signaling pathway in mouse nasal and tracheal epithelial cells

Objectives

Our purpose was to investigate the role of the nitric oxide (NO) signaling pathway in the regulation of ciliary beat frequency (CBF) in mouse nasal and tracheal epithelial cells.

Methods

We studied the effects of the NO donor l-arginine (L-Arg) and specific inhibitors of the NO signaling pathway on CBF of both nasal and tracheal epithelial cells by using high-speed digital microscopy. We also examined eNOS, sGC β, PKG I and acetylated α tubulin expression in native mouse nasal and tracheal epithelium using immunohistochemical methods.

Results

L-Arg significantly increased CBF of cultured nasal and tracheal epithelial cells, and the effects were blocked by pretreatment with N^G-nitro-l-arginine methyl ester (L-NAME), a NOS inhibitor, with LY-83583, a sGC inhibitor, or with KT-5823, a PKG inhibitor. Positive immunostaining for NO signaling molecules including eNOS, sGC β and PKG I was observed in either nasal or tracheal ciliated epithelium.

Conclusion

NO plays a role in regulating CBF of mouse respiratory epithelial cells via a eNOS–NO–sGC β–cGMP–PKG I pathway.     

Insulin-like actions of vanadate are mediated in an insulin-receptor-independent manner via non-receptor protein tyrosine kinases and protein phosphotyrosine phosphatases

Most or all mammalian cells contain vanadium at a concentration of 0.1–1.0 M. The bulk of the vanadium in cells is probably in the reduced vanadyl (IV) form. Although this element is essential and should be present in the diet in minute quantities, no known physiological role for vanadium has been found thus far. In the years 1975–1980 the vanadate ion was shown to act as an efficient inhibitor of Na⁺,K⁺-ATPase and of other related phosphohydrolyzes as well. In 1980 it was observed that vanadate vanadyl, when added to intact rat adipocytes, mimics the biological actions of insulin in stimulating hexose uptake and glucose oxidation. This initiated a long, currently active, field of research among basic scientists and diabetologists. Several of the aspects studied are reviewed here.     

Roles of pH and cyclic adenosine monophosphate in the acquisition of potential for sperm motility during migration from the sea to the river in chum salmon

In the natural process of the migration of chum salmon from the sea to the river, spermatozoa moved from the testis to the sperm duct, and the pH value of seminal plasma, concentration of cyclic adenosine monophosphate (AMP) in the sperm cells, and potential for sperm motility increased. Cyclic AMP levels and the potential for motility gradually increased when testis spermatozoa with no capacity for movement were incubated in the artificial seminal plasma of which the pH was much the same as, or higher than, the pH of natural seminal plasma from the sperm duct. Such correlation in motility, pH, and cyclic AMP suggests that the increases in seminal pH and intracellular cyclic AMP level during passage of spermatozoa from the testis to the sperm duct cause the acquisition of potential for motility. Motility of testicular spermatozoa demembranated with Triton X-100 was very low in fish caught in the sea, while motility of spermatozoa from the posterior portion of the sperm duct was much higher in fish caught in the river. Furthermore, nondemembranated, intact spermatozoa showed a lag in the timing of the acquisition of potential for motility vs. demembranated spermatozoa: The demembranated sperm exhibited the potential earlier than the nondemembranated sperm. These data suggest that increase in activity of the motile apparatus, the axoneme, is a prerequisite, in part, for the acquisition of sperm motility, whereas the development of some function of the plasma membrane also contributes to this phenomenon. © 1993 Wiley-Liss, Inc.