Microtubule reorganization during mitotic cell division in the dinoflagellate Ostreospis cf. ovata.

Dinoflagellates are marine organisms that undergo seasonal proliferation events known as algal blooms. Vegetative cell proliferation is a main contributing factor in these events. However, mechanistical understanding of mitosis and cytokinesis in dinoflagellates remains rudimentary. Using an optimized immunofluorescence protocol, we analysed changes in microtubule organization occurring during the mitotic cycle of the toxic dinoflagellate Ostreopsis cf. ovata. We find that the flagella and the cortical microtubule array persist throughout the mitotic cycle. Two cytoplasmic microtubule bundles originate from the ventral area, where the basal bodies are located - a cortical bundle and a cytoplasmic bundle. The latter associates with the nucleus in the cell centre before mitosis and with the acentrosomal extranuclear spindle during mitosis. Analysis of tubulin post-translational modifications identifies two populations of spindle microtubules - polar acetylated microtubules, whose length is constant, and central tyrosinated microtubules, which elongate during chromosome segregation. During cell division a microtubule-rich structure forms along the dorsal-ventral axis, associated with the site of cytokinesis, consistent with a cytokinetic mechanism that is independent of the actomyosin ring typical of animal and yeast cells.

Novel cytoskeletal traits in the intestinal parasites (Squirmida, Platyproteum vivax) of Pacific peanut worms (Sipuncula, Phascolosoma agassizii).

The cytoskeletal organization of a squirmid, namely Platyproteum vivax, was investigated with confocal laser scanning microscopy (CLSM) to refine inferences about convergent evolution among intestinal parasites of marine invertebrates. Platyproteum inhabits Pacific peanut worms (Phascolosoma agassizii) and has traits that are similar to other lineages of myzozoan parasites, namely gregarine apicomplexans within Selenidium, such as conspicuous feeding stages, called "trophozoites," capable of dynamic undulations. SEM and CLSM of P. vivax revealed an inconspicuous flagellar apparatus and a uniform array of longitudinal microtubules organized in bundles (LMBs). Extreme flattening of the trophozoites and a consistently oblique morphology of the anterior end provided a reliable way to distinguish dorsal and ventral surfaces. CLSM revealed a novel system of microtubules oriented in the flattened dorsoventral plane. Most of these dorsoventral microtubule bundles (DVMBs) had a punctate distribution and were evenly spaced along a curved line spanning the longitudinal axis of the trophozoites. This configuration of microtubules is inferred to function in maintaining the flattened shape of the trophozoites and facilitate dynamic undulations. The novel traits in Platyproteum are consistent with phylogenomic data showing that this lineage is only distantly related to Selenidium and other marine gregarine apicomplexans with dynamic intestinal trophozoites.

Tetrahymena Poc5 is a transient basal body component that is important for basal body maturation.

Basal bodies (BBs) are microtubule-based organelles that act as a template for and stabilize cilia at the cell surface. Centrins ubiquitously associate with BBs and function in BB assembly, maturation and stability. Human POC5 (hPOC5) is a highly conserved centrin-binding protein that binds centrins through Sfi1p-like repeats and is required for building full-length, mature centrioles. Here, we use the BB-rich cytoskeleton of Tetrahymena thermophila to characterize Poc5 BB functions. Tetrahymena Poc5 (TtPoc5) uniquely incorporates into assembling BBs and is then removed from mature BBs prior to ciliogenesis. Complete genomic knockout of TtPOC5 leads to a significantly increased production of BBs, yet a markedly reduced ciliary density, both of which are rescued by reintroduction of TtPoc5. A second Tetrahymena POC5-like gene, SFR1, is similarly implicated in modulating BB production. When TtPOC5 and SFR1 are co-deleted, cell viability is compromised and BB overproduction is exacerbated. Overproduced BBs display defective transition zone formation and a diminished capacity for ciliogenesis. This study uncovers a requirement for Poc5 in building mature BBs, providing a possible functional link between hPOC5 mutations and impaired cilia.This article has an associated First Person interview with the first author of the paper.

The interplay of self-assembly and target binding in centrin 1 from Toxoplasma gondii.

Centrins are conserved calcium (Ca2+)-binding proteins typically associated with centrosomes that have been implicated in several biological processes. In Toxoplasma gondii, a parasite that causes toxoplasmosis, three centrin isoforms have been recognized. We have recently characterized the metal binding and structural features of isoform 1 (TgCEN1), demonstrating that it possesses properties consistent with a role as a Ca2+ sensor and displays a Ca2+-dependent tendency to self-assemble. Herein, we expanded our studies, focusing on the self-association and target binding properties of TgCEN1 by combining biophysical techniques including dynamic light scattering, isothermal titration calorimetry, nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy. We found that the self-assembly process of TgCEN1 depends on different physicochemical factors, including Ca2+ concentration, temperature, and protein concentration, and is mediated by both electrostatic and hydrophobic interactions. The process is completely abolished upon removal of the first 21-residues of the protein and is significantly reduced in the presence of a binding target peptide derived from the human XPC protein (P17-XPC). Titration of P17-XPC to the intact protein and isolated domains showed that TgCEN1 possesses two binding sites with distinct affinities and Ca2+ sensitivity; a high-affinity site in the C-lobe which may be constitutively bound to the peptide and a low-affinity site in the N-lobe which is active only upon Ca2+ stimulus. Overall, our results suggest a specific mechanism of TgCEN1 for Ca2+-modulated target binding and support a N-to-C self-assembly mode, in which the first 21-residues of one molecule likely interact with the C-lobe of the other.

The roles of Centrin 2 and Dynein Light Chain 8a in apical secretory organelles discharge of Toxoplasma gondii.

To efficiently enter host cells, apicomplexan parasites such as Toxoplasma gondii rely on an apical complex composed of tubulin-based structures as well as two sets of secretory organelles named micronemes and rhoptries. The trafficking and docking of these organelles to the apical pole of the parasite is crucial for the discharge of their contents. Here, we describe two proteins typically associated with microtubules, Centrin 2 (CEN2) and Dynein Light Chain 8a (DLC8a), that are required for efficient host cell invasion. CEN2 localizes to four different compartments, and remarkably, conditional depletion of the protein occurs in stepwise manner, sequentially depleting the protein pools from each location. This phenomenon allowed us to discern the essential function of the apical pool of CEN2 for microneme secretion, motility, invasion and egress. DLC8a localizes to the conoid, and its depletion also perturbs microneme exocytosis in addition to the apical docking of the rhoptry organelles, causing a severe defect in host cell invasion. Phenotypic characterization of CEN2 and DLC8a indicates that while both proteins participate in microneme secretion, they likely act at different steps along the cascade of events leading to organelle exocytosis.

Maintenance of hook complex integrity and centrin arm assembly facilitates flagellum inheritance in Trypanosoma brucei.

Inheritance of the newly assembled flagellum in the human parasite Trypanosoma brucei depends on the faithful duplication and segregation of multiple flagellum-associated cytoskeletal structures, including the hook complex and its associated centrin arm. The biological functions of this unique hook complex-centrin arm assembly remain poorly understood. Here, we report a hook complex-associated protein named BOH2 that plays an essential role in promoting flagellum inheritance. BOH2 localizes to the hooked part of the hook complex by bridging the hook complex, the centrin arm, and the flagellum attachment zone filament. Depletion of BOH2 caused the loss of the shank part of the hook complex and its associated protein TbSmee1, disrupted the assembly of the centrin arm and the recruitment of centrin arm-associated protein CAAP1, inhibited the assembly of the flagellum attachment zone, and caused flagellum mispositioning and detachment. These results demonstrate crucial roles of BOH2 in maintaining hook complex integrity and promoting centrin arm formation and suggest that proper assembly of the hook complex-centrin arm structure facilitates flagellum inheritance.

Centrin2 from the human parasite Toxoplasma gondii is required for its invasion and intracellular replication.

Centrins are EF-hand containing proteins ubiquitously found in eukaryotes and are key components of centrioles/basal bodies as well as certain contractile fibers. We previously identified three centrins in the human parasite Toxoplasma gondii, all of which localized to the centrioles. However, one of them, T. gondii (Tg) Centrin2 (CEN2), is also targeted to structures at the apical and basal ends of the parasite, as well as to annuli at the base of the apical cap of the membrane cortex. The role(s) that CEN2 play in these locations were unknown. Here, we report the functional characterization of CEN2 using a conditional knockdown method that combines transcriptional and protein stability control. The knockdown resulted in an ordered loss of CEN2 from its four compartments, due to differences in incorporation kinetics and structural inheritance over successive generations. This was correlated with a major invasion deficiency at early stages of CEN2 knockdown, and replication defects at later stages. These results indicate that CEN2 is incorporated into multiple cytoskeletal structures to serve distinct functions that are required for parasite survival.

Differential requirements for the EF-hand domains of human centrin 2 in primary ciliogenesis and nucleotide excision repair.

Centrin 2 is a small conserved calcium-binding protein that localizes to the centriolar distal lumen in human cells. It is required for efficient primary ciliogenesis and nucleotide excision repair (NER). Centrin 2 forms part of the xeroderma pigmentosum group C protein complex. To explore how centrin 2 contributes to these distinct processes, we mutated the four calcium-binding EF-hand domains of human centrin 2. Centrin 2 in which all four EF-hands had been mutated to ablate calcium binding (4DA mutant) was capable of supporting in vitro NER and was as effective as the wild-type protein in rescuing the UV sensitivity of centrin 2-null cells. However, we found that mutation of any of the EF-hand domains impaired primary ciliogenesis in human TERT-RPE1 cells to the same extent as deletion of centrin 2. Phenotypic analysis of the 4DA mutant revealed defects in centrosome localization, centriole satellite assembly, ciliary assembly and function and in interactions with POC5 and SFI1. These observations indicate that centrin 2 requires calcium-binding capacity for its primary ciliogenesis functions, but not for NER, and suggest that these functions require centrin 2 to be capable of forming complexes with partner proteins.This article has an associated First Person interview with the first author of the paper.

Conformational scanning of individual EF-hand motifs of calcium sensor protein centrin-1.

Centrin-1, a Ca2+ sensor protein of the centrin family is a crucial player for cell division in eukaryotes and plays a key role in the microtubule organising centre. Despite being regarded as a calcium sensor with a matched structure to calmodulin/troponin C, the protein undergoes mild changes in conformation and binds Ca2+ with moderate affinity. We present an in-depth analysis of the Ca2+ sensing by individual EF-hand motifs of centrin-1 and address unsolved questions of the rationales for moderate affinity and conformational transitions of the protein. Employing the more sensitive approach of Trp scanning of individual EF-hand motif, we have undertaken an exhaustive investigation of Ca2+ binding to individual EF-hand motifs, named EF1 to EF4. All four EF-hand motifs of centrin-1 are structural as all of them bind both Ca2+ and Mg2+. EF1 and EF4 are the most flexible sites as they undergo drastic conformational changes following Ca2+ binding, whereas EF3 responds to Ca2+ minimally. On the other hand, EF2 moves towards the protein surface upon binding Ca2+. The independent filling mode of Ca2+ to EF-hand motifs and lack of intermotif communication explain the lack of cooperativity of binding, thus constraining centrin-1 to a moderate affinity binding protein. Thus, centrin-1 is distinct from other calcium sensors such as calmodulin.

Insights into the Morphology of Haplozoan Parasites (Dinoflagellata) using Confocal Laser Scanning Microscopy.

We describe new insights into the morphology and life history of the bizarre parasite Haplozoon axiothellae (Dinoflagellata) using light microscopy (LM), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Trophonts were isolated from the intestines of host maldanid polychaetes, Axiothella rubrocincta, collected from San Juan Island, Washington, USA. LM and SEM confirmed features previously observed, such as amphiesmal projections, mature and immature junctions between the nucleated compartments of the vermiform syncytium and visible polygonal alveoli. CLSM of adult trophonts fluorescently stained for DNA, tubulin, centrin, and plasma membrane demonstrated several new ultrastructural traits: (1) an extensive basket of parallel microtubules within the trophomere used for host attachment, (2) two physically separated MTOCs (i.e. putative pairs of basal bodies) beneath pores on the ventral side of each compartment, (3) robust mitotic and/or meiotic spindles associated with one to four nuclei in each compartment, (4) spindles with polar bodies that are disconnected from the MTOCs, (5) a centrin-stained fibril within the trophomeres that potentially functions to retract the motile stylet, and (6) cytokinesis in the posterior-most compartments. This study renames haplozoan compartments using the suffix "-mere" rather than "-cyte" (i.e. trophomere, gonomere, sporomere) to reflect their status within a single syncytium.

Expression analysis of centrin gene in promastigote and amastigote forms of leishmania infantum iranian isolates: a promising target for live attenuated vaccine development against canine leishmaniasis.

BACKGROUND: Leishmania parasites express various essential proteins in different growth phases (logarithmic/stationary) and forms (promastigote/amastigote). Targeting the genes encoding such proteins paves the way for controlling these parasites. Centrin is an essential gene, which its protein product seems to be vital for the proliferation of Leishmania parasites. Herein, this study was contrived to analyze the expression level of the centrin gene in different growth phases and forms of Leishmania infantum (L. infantum) parasites isolated from various endemic areas of canine leishmaniasis (CanL) in Iran. RESULTS: All three collected isolates were identified as L. infantum using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Real-time reverse transcription (RT)-PCR revealed a statistically significant up-regulation (3.13-fold) in the logarithmic phase promastigotes compared to stationary ones (p < 0.01), whereas centrin was expressed equally in intracellular amastigotes at different time points during cell culture. Also, our finding revealed a slight up-regulation of the centrin gene (1.22-fold) in the intracellular amastigotes compared to logarithmic phase promastigotes, which was found statistically non-significant (p > 0.05). CONCLUSIONS: Centrin gene in Iranian isolates of L. infantum is more expressed in exponential than stationary phases and seems to be considered as a promising target in the development of a genetically modified live attenuated vaccine for CanL control.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana.

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp-CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein-peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

Centriolar defects, centrin 1 alterations, and FISH studies in human spermatozoa of a male partner of a couple that produces aneuploid embryos in natural and artificial fertilization.

PURPOSE: To study the potential paternal contribution to aneuploidies in the man of a couple who obtained trisomic embryos with natural and assisted fertilization. METHODS: Semen analysis, immunofluorescence for localization of tubulin and centrin 1, transmission electron microscopy (TEM), and fluorescence in situ hybridization (FISH) analysis for chromosomes 18 and 9 were performed. Sperm of fertile men were used as controls. RESULTS: The percentages of sperm motility and normal forms were decreased. The percentages of sperm with tail reduced in dimension, headless tails, coiled tails, and altered head-tail junction were significantly higher (P < 0.01) in the patient than in controls, whereas the percentage of sperm with a normal centrin 1 localization (two spots in the centriolar area) was significantly reduced (P < 0.01) in the patient. Immunofluorescence with anti-tubulin antibody showed that in most of the patient's sperm connecting pieces (83.00 ± 1.78%), two spots were present, indicating prominent proximal centriole/centriolar adjunct and evident distal centriole, whereas controls' sperm displayed a single spot, indicating the proximal centriole. The percentage of sperm with two spots was significantly higher (P < 0.01) in the patient than in controls. TEM analysis showed that centriolar adjuncts of the patient's sperm were significantly longer (721.80 ± 122.26 nm) than in controls' sperm (310.00 ± 64.11 nm; P < 0.001). The aneuploidy frequencies of the patient's sperm, detected by FISH analysis, were increased with respect to controls. CONCLUSION: A paternal contribution to sperm aneuploidies cannot be excluded since the patient's sperm showed altered morphology, immature centriolar adjunct, presence of evident distal centriole, scarce presence of centrin 1, and high aneuploidy frequency.

Structural Basis for the Functional Diversity of Centrins: A Focus on Calcium Sensing Properties and Target Recognition.

Centrins are a family of small, EF hand-containing proteins that are found in all eukaryotes and are often complexed with centrosome-related structures. Since their discovery, centrins have attracted increasing interest due to their multiple, diverse cellular functions. Centrins are similar to calmodulin (CaM) in size, structure and domain organization, although in contrast to CaM, the majority of centrins possess at least one calcium (Ca2+) binding site that is non-functional, thus displaying large variance in Ca2+ sensing abilities that could support their functional versatility. In this review, we summarize current knowledge on centrins from both biophysical and structural perspectives with an emphasis on centrin-target interactions. In-depth analysis of the Ca2+ sensing properties of centrins and structures of centrins complexed with target proteins can provide useful insight into the mechanisms of the different functions of centrins and how these proteins contribute to the complexity of the Ca2+ signaling cascade. Moreover, it can help to better understand the functional redundancy of centrin isoforms and centrin-binding proteins.

Deletion of both centrin 2 (CETN2) and CETN3 destabilizes the distal connecting cilium of mouse photoreceptors.

Centrins (CETN1-4) are ubiquitous and conserved EF-hand-family Ca2+-binding proteins associated with the centrosome, basal body, and transition zone. Deletion of CETN1 or CETN2 in mice causes male infertility or dysosmia, respectively, without affecting photoreceptor function. However, it remains unclear to what extent centrins are redundant with each other in photoreceptors. Here, to explore centrin redundancy, we generated Cetn3GT/GT single-knockout and Cetn2-/-;Cetn3GT/GT double-knockout mice. Whereas the Cetn3 deletion alone did not affect photoreceptor function, simultaneous ablation of Cetn2 and Cetn3 resulted in attenuated scotopic and photopic electroretinography (ERG) responses in mice at 3 months of age, with nearly complete retina degeneration at 1 year. Removal of CETN2 and CETN3 activity from the lumen of the connecting cilium (CC) destabilized the photoreceptor axoneme and reduced the CC length as early as postnatal day 22 (P22). In Cetn2-/-;Cetn3GT/GT double-knockout mice, spermatogenesis-associated 7 (SPATA7), a key organizer of the photoreceptor-specific distal CC, was depleted gradually, and CETN1 was condensed to the mid-segment of the CC. Ultrastructural analysis revealed that in this double knockout, the axoneme of the CC expanded radially at the distal end, with vertically misaligned outer segment discs and membrane whorls. These observations suggest that CETN2 and CETN3 cooperate in stabilizing the CC/axoneme structure.

Characterisation of three systematic sperm tail defects and their influence on ICSI outcome.

This study characterized three cases of systematic sperm tail defects using electron microscopy and immunolocalisation of centrin 1 and tubulin and explored their impact on ICSI outcome. Structural sperm tail defects of possible genetic origin were suspected as the eosin test revealed a sperm viability of >70% despite severe asthenozoospermia or the absence of motility. In Patient 1, 80%-85% of axoneme cross sections was incomplete. The fluorescent signal of tubulin was weak along the entire tail; the signal of centrin 1 was normal. After ICSI, a female healthy baby was born. Patient 2 showed spermatozoa with tails reduced in length at different levels, axonemal and periaxonemal alterations and fragility of head-tail junction. Centrin 1 was altered in 80% of sperm. After ICSI, no embryos were obtained. Patient 3 showed tails reduced in length at light and fluorescence microscopy; ultrastructural study revealed a condition of dysplasia of fibrous sheath with heterogeneity of tails' length. The signal for centrin 1 was altered in 50% of spermatozoa; two embryos were transferred without pregnancy. The correct diagnosis of sperm pathology is important in case of systematic sperm defects as it enables the clinician to improve patient's management and to provide an adequate genetic counselling.

New insights into the interaction of centrin with Sfi1.

Centrin binds to Rad4(XPC) and Sfi1 through the hydrophobic motif W(1)xxL(4)xxxL(8) in the opposite orientation. Rad4 has one motif, but Sfi1 has approximately 20 repeats, each of which interacts with a centrin molecule. To investigate the parameters involved in centrin binding, we purified a ScSfi1 domain containing 6 repeats complexed with either yeast centrin Cdc31 or human centrin 1. The present study was performed using mutagenesis of centrin and of Sfi1 residues involved in centrin binding and the stability of the centrin-centrin complexes was assessed using thermal denaturation and CD. Calcium stabilized these complexes, as indicated by the Tm increases measured by circular dichroism. The complexes, which were composed of Sfi1 variants and yeast centrin, were analysed in the presence of EDTA. The replacement of W with F within the repeat region yielded a functional repeat (Tm 45°C). The replacement of W with A in two adjacent Sfi1 repeats reduced the thermal stability of the Sfi1-centrin complexes (40°C). We analysed three HsCen1 variants that were homologous to the yeast mutants and induced cell cycle arrest during the G2/M transition. The HsCen1 variants E105K and F113L reduced the thermal stability (50°C, 50°C) of the ScSfi1-HsCen1 complexes; in contrast, the A109T variant exhibited no change in thermal stability relative to the wild-type (60°C). Conversely to ScCdc31, there were no apparent centrin-centrin interactions with wild-type HsCen1, but they did occur for the S170D mutation that mimics PKA phosphorylation at the S170 residue.

Sperm with fibrous sheath dysplasia and anomalies in head-neck junction: focus on centriole and centrin 1.

Spermatozoa with a rare combination of two monomorphic sperm defects, dysplasia of the fibrous sheath (DFS) and alterations in head-mid-piece junction were analysed. The main focus was to explore the status of the centriole, a key organisation during fertilisation, using the centrin 1, a calcium-binding protein linked to this structure. The sperm quality was examined by light, scanning and transmission electron microscopy (SEM, TEM); immunocytochemistry was performed for tubulin, A-kinase anchor protein 4 (AKAP4) and centrin 1. Spermatozoa showed DFS defect associated with anomalies in head-tail attachment detected by SEM and TEM. Immunolocalisation of tubulin, AKAP4 and centrin 1 confirmed these alterations. Centrin 1 was visible in 67% of spermatozoa (in only 13% centrin localised in a normal position); in the majority of sperm centrin 1's location was altered, sometimes bent; often four spots, indicating the presence of two implantation fossae, were detected. At the centriolar level, immunoreactive fragments, frequently invading the entire short and thick tail, were observed. Centrin 1 is an essential component of the spermatozoa connecting piece and plays a role in centrosome dynamics during sperm morphogenesis and in zygotes and early embryos during spindle assembly. It is important to shed light on these rare conditions in order to better manage the patients during assisted reproductive technology.

Generation and Characterization of a Tissue-Specific Centrosome Indicator Mouse Line.

Centrosomes are major microtubule organizing centers (MTOCs) that play an important role in chromosome segregation during cell division. Centrosomes provide a stable anchor for microtubules, constituting the centers of the spindle poles in mitotic cells, and determining the orientation of cell division. However, visualization of centrosomes is challenging because of their small size. Especially in mouse tissues, it has been extremely challenging to observe centrosomes belonging to a specific cell type of interest among multiple comingled cell types. To overcome this obstacle, we generated a tissue-specific centrosome indicator. In this mouse line, a construct containing a floxed neomyocin resistance gene with a triplicate polyA sequence followed by an EGFP-Centrin1 fusion cassette was knocked into the Rosa locus. Upon Cre-mediated excision, EGFP-Centrin1 was expressed under the control of the Rosa locus. Experiments utilizing mouse embryo fibroblasts (MEFs) demonstrated the feasibility of real-time imaging, and showed that EGFP-Centrin1 expression mirrored the endogenous centrosome cycle, undergoing precisely one round of duplication through the cell cycle. Moreover, experiments using embryo and adult mouse tissues demonstrated that EGFP-Centrin1 specifically mirrors the localization of endogenous centrosomes. genesis 54:286-296, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.

Optimization of purification method and characterization of recombinant human Centrin-1.

Centrins are acidic proteins, present in all eukaryotes to perform imperative roles in centrosome positioning and segregation. Existing methods for the purification of centrins for biophysical studies involves either multiple steps or yields protein with an affinity tag, which pins additional tag-cleavage step. Therefore, we have made an attempt to develop a simple and single step method for protein purification. We have performed categorical evaluation of existing methods, and describe a one-step procedure based on cleavable Intein-tag, which can be utilized for routine preparation of any isoform of centrins. Since human Centrin-1 and Centrin-2 are devoid of Trp, we exploit this feature to assess the purity of the protein using Tyr fluorescence; an essential point ignored generally. In addition, we report important spectral and hydrodynamic characteristics of human Centrin-1, accounting that HsCentrin-1 has moderate affinity for Ca(2+). Centrin-1 does not gain structure as seen by far- and near-UV circular dichroism, rather there is a loss of ellipticity, though inconsiderable upon binding Ca(2+).