Precocious cleavage furrows simultaneously move and ingress when kinetochore microtubules are depolymerized in <Emphasis Type="Italic">Mesostoma ehrenbergii</Emphasis> spermatocytes

A “precocious” cleavage furrow develops and ingresses during early prometaphase in Mesostoma ehrenbergii spermatocytes (Forer and Pickett-Heaps Eur J Cell Biol 89:607-618, 2010). In response to chromosome movements which regularly occur during prometaphase and that alter the balance of chromosomes in the two half-spindles, the precocious furrow shifts its position along the cell, moving 2–3 μm towards the half cell with fewer chromosomes (Ferraro-Gideon et al. Cell Biol Int 37:892-898, 2013). This process continues until proper segregation is achieved and the cell enters anaphase with the cleavage furrow again in the middle of the cell. At anaphase, the furrow recommences ingression. Spindle microtubules (MTs) are implicated in various furrow positioning models, and our experiments studied the responses of the precocious furrows to the absence of spindle MTs. We depolymerized spindle MTs during prometaphase using various concentrations of nocodazole (NOC) and colcemid. The expected result is that the furrow should regress and chromosomes remain in the midzone of the cell (Cassimeris et al. J Cell Sci 96:9-15, 1990). Instead, the furrows commenced ingression and all three bivalent chromosomes moved to one pole while the univalent chromosomes, that usually reside at the two poles, either remained at their poles or moved to the opposite pole along with the bivalents, as described elsewhere (Fegaras and Forer 2018). The microtubules were completely depolymerized by the drugs, as indicated by immunofluorescence staining of treated cells (Fegaras and Forer 2018), and in the absence of microtubules, the furrows often ingressed (in 33/61 cells) at a rate similar to normal anaphase ingression (~?1 μm/min), while often simultaneously moving toward one pole. Thus, these results indicate that in the absence of anaphase and of spindle microtubules, cleavage furrows resume ingression.     

Endomitotic Megakaryocytes that Form a Bipolar Spindle Exhibit Cleavage Furrow Ingression Followed by Furrow Regression

Megakaryocyte (MK) differentiation is marked by the development of progressive polyploidy, due to repeated incomplete cell cycles in which mitosis is aborted during anaphase, a process termed endomitosis. We have postulated that anaphase in endomitotic MKs diverges from diploid mitosis at a point distal to the assembly of the midzone, possibly involving impaired cleavage furrow progression. To define the extent of furrow initiation and ingression in endomitosis, we performed time-lapse imaging of MKs expressing yellow fluorescent protein (YFP)-tubulin and monitored shape change as they progressed through anaphase. We found that in early endomitotic cells that have a bipolar spindle, cleavage furrows form that can undergo significant ingression, but furrows regress to produce polyploid cells. Compared to cells that divide, cells that exhibit furrow regression have a slower rate of furrow ingression and do not furrow as deeply. More highly polyploid MKs undergoing additional endomitotic cycles also show measurable furrowing that is followed by regression, but the magnitude of the shape change is less than seen in the early MKs. This suggests that in the earliest endomitotic cycles when there is formation of a bipolar spindle, the failure of cytokinesis occurs late, following assembly and initial constriction of the actin/myosin ring, whereas in endomitotic MKs that are already polyploid there is secondary inhibition of furrow progression. This behavior of furrow ingression followed by regression may explain why midbody remnants are occasionally observed in polyploid MKs. This finding has important implications for the potential mechanisms for cytokinesis failure in endomitosis.     

Polo-like kinase 1 triggers the initiation of cytokinesis in human cells by promoting recruitment of the RhoGEF Ect2 to the central spindle

Cytokinesis of animal cells requires ingression of the actomyosin-based contractile ring between segregated sister genomes. Localization of the RhoGEF Ect2 to the central spindle at anaphase promotes local activation of the RhoA GTPase, which induces assembly and ingression of the contractile ring. Here we have used BI 2536, an inhibitor of the mitotic kinase Plk1, to analyze the functions of this enzyme during late mitosis in human cells. We show that Plk1 acts after Cdk1 inactivation and independently from Aurora B to promote RhoA accumulation at the equator, contractile ring formation, and cleavage furrow ingression. Inhibition of Plk1 abolishes the interaction of Ect2 with its activator and midzone anchor, HsCyk-4, thereby preventing localization of Ect2 to the central spindle. We propose that late mitotic Plk1 activity promotes recruitment of Ect2 to the central spindle, triggering the initiation of cytokinesis and contributing to cleavage plane specification in human cells.     

Augmin shapes the anaphase spindle for efficient cytokinetic furrow ingression and abscission

During anaphase, distinct populations of microtubules (MTs) form by either centrosome-dependent or augmin-dependent nucleation. It remains largely unknown whether these different MT populations contribute distinct functions to cytokinesis. Here we show that augmin-dependent MTs are required for the progression of both furrow ingression and abscission. Augmin depletion reduced the accumulation of anillin, a contractile ring regulator at the cell equator, yet centrosomal MTs were sufficient to mediate RhoA activation at the furrow. This defect in contractile ring organization, combined with incomplete spindle pole separation during anaphase, led to impaired furrow ingression. During the late stages of cytokinesis, astral MTs formed bundles in the intercellular bridge, but these failed to assemble a focused midbody structure and did not establish tight linkage to the plasma membrane, resulting in furrow regression. Thus augmin-dependent acentrosomal MTs and centrosomal MTs contribute to nonredundant targeting mechanisms of different cytokinesis factors, which are required for the formation of a functional contractile ring and midbody.     

Study of folliculogenesis in vivo in guinea pig

Understanding normal folliculogenesis in guinea pigs is fundamental as a first step towards the development of a guinea pig follicle culture system. The aims of this study were (1) to characterise morphological changes during follicular development in vivo and (2) to describe the growth pattern of follicles. Cycling guinea pigs were infused with 5-bromo-2′-deoxyuridine for 1 or 2 weeks and sacrificed at time points ranging from 0 to 37 days after the infusion. The granulosa cell number in the largest cross-sections increased from 25.0 ± 6.1 (mean ± S.D.) in primary (type 2) to 192.0 ± 65.9 in preantral (type 5) and 256.3 ± 96.9 in antral (type 6) follicles. The oocyte diameter increased from 44.8 ± 6.2 μm (type 2) to 72.8 ± 9.1 μm (type 5) and 78.9 ± 9.3 μm (type 6) and the follicle diameter from 67.9 ± 10.1 μm (type 2) to 188.9 ± 29.7 μm (type 5) and 231.0 ± 56.1 μm (type 6). After a 1-week labelling period, about 71% of type 2 follicles had at least one labelled granulosa cell, as did 95% of type 3–4, and 100% of type 5 and 6. About 1 week was needed to achieve 95% mitotic activity in granulosa cells (GC) of type 5 and 6 follicles, while about 2 weeks was required to achieve 100% mitotic activity in GC of type 3–4 and more than 2 weeks for GC of type 2 follicles. These data provide some baselines for the examination of a guinea pig follicle culture system.     

Changes in the size distribution of goat steroidogenic luteal cells during pregnancy

Experiments were conducted to investigate the size distribution of goat steroidogenic luteal cells throughout pregnancy. Corpora lutea were collected from very early (<6 weeks), early (6–8 weeks), middle (9–14 weeks) or late (15–18 weeks) stages of pregnancy. Luteal tissue was dissociated into single-cell suspension by enzyme treatments. Cells were stained for 3β-hydroxysteroid dehydrogenase (3β-HSD) activity, a marker for steroidogenic cells. The steroidogenic cells covered a wide spectrum of size ranging from 5 to 45 μm in diameter. There was a significant increase in mean cell diameter (P>0.01) as pregnancy progressed. Mean diameter of 3β-HSD positive cells increased from 14.73±0.35 μm in the corpus luteum of very early pregnancy to 24.20±0.45 μm in the corpus luteum of late pregnancy. The ratio of large (>20 μm in diameter) to small (5–20 μm in diameter) luteal cells was 0.28:1.0 in very early pregnancy, with the 7.5–15 μm cell size class being dominant. However, the ratio of large-to-small luteal cells was increased to 1.77:1.0 μm as pregnancy advanced and 25–35 μm cell sizes became predominant. It is likely that small luteal cells could develop into large cells as pregnancy progresses. Development of pregnancy is also associated with an increase in size of steroidogenic luteal cells.     

2,2′-Dihydroxybenzophenones and their carbonyl N-analogues as inhibitor scaffolds for MDR-involved human glutathione transferase isoenzyme A1-1

The MDR-involved human GSTA1-1, an important isoenzyme overexpressed in several tumors leading to chemotherapeutic-resistant tumour cells, has been targeted by 2,2′-dihydroxybenzophenones and some of their carbonyl N-analogues, as its potential inhibitors. A structure-based library of the latter was built-up by a nucleophilic cleavage of suitably substituted xanthones to 2,2′-dihydroxy-benzophenones (5–9) and subsequent formation of their N-derivatives (oximes 11–13 and N-acyl hydrazones 14–16). Screening against hGSTA1-1 led to benzophenones 6 and 8, and hydrazones 14 and 16, having the highest inhibition potency (IC₅₀ values in the range 0.18 ± 0.02 to 1.77 ± 0.10 μM). Enzyme inhibition kinetics, molecular modeling and docking studies showed that they interact primarily at the CDNB-binding catalytic site of the enzyme. In addition, the results from cytotoxicity studies with human colon adenocarcinoma cells showed low LC₅₀ values for benzophenone 6 and its N-acyl hydrazone analogue 14 (31.4 ± 0.4 μM and 87 ± 1.9 μM, respectively), in addition to the strong enzyme inhibition profile (IC₅₀₍₆₎ = 1,77 ± 0.10 μM; IC₅₀₍₁₄₎ = 0.33 ± 0.05 μM). These structures may serve as leads for the design of new potent mono- and bi-functional inhibitors and pro-drugs against human GTSs.     

Anillin is a substrate of anaphase-promoting complex/cyclosome (APC/C) that controls spatial contractility of myosin during late cytokinesis

Anillin, an actin-binding protein localized at the cleavage furrow, is required for cytokinesis. Through an in vitro expression screen, we identified anillin as a substrate of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. We found that the levels of anillin fluctuate in the cell cycle, peaking in mitosis and dropping drastically during mitotic exit. Ubiquitination of anillin required a destruction-box and was mediated by Cdh1, an activator of APC/C. Overexpression of Cdh1 reduced the levels of anillin, whereas inactivation of APC/C(Cdh1) increased the half-life of anillin. Functionally, anillin was required for the completion of cytokinesis. In anillin knockdown cells, the cleavage furrow ingressed but failed to complete the ingression. At late cytokinesis, the cytosol and DNA in knockdown cells underwent rapid myosin-based oscillatory movement across the furrow. During this movement, RhoA and active myosin were absent from the cleavage furrow, and myosin was redistributed to cortical patches, which powers the random oscillatory movement. We concluded that anillin functions to maintain the localization of active myosin, thereby ensuring the spatial control of concerted contraction during cytokinesis.     

Discovery and characterisation of hydrazines as inhibitors of the immune suppressive enzyme,indoleamine 2,3-dioxygenase 1 (IDO1)

Screening of a fragment library identified 2-hydrazinobenzothiazole as a potent inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme expressed by tumours that suppresses the immune system. Spectroscopic studies indicated that 2-hydrazinobenzothiazole interacted with the IDO1 haem and in silico docking predicted that the interaction was through hydrazine. Subsequent studies of hydrazine derivatives identified phenylhydrazine (IC₅₀ = 0.25 ± 0.07 μM) to be 32-fold more potent than 2-hydrazinobenzothiazole (IC₅₀ = 8.0 ± 2.3 μM) in inhibiting rhIDO1 and that it inhibited cellular IDO1 at concentrations that were noncytotoxic to cells. Here, phenylhydrazine is shown to inhibit IDO1 through binding to haem.     

Pentapeptide boronic acid inhibitors of Mycobacterium tuberculosis MycP1 protease

Mycosin protease-1 (MycP₁) cleaves ESX secretion-associated protein B (EspB) that is a virulence factor of Mycobacterium tuberculosis, and accommodates an octapeptide, AVKAASLG, as a short peptide substrate. Because peptidoboronic acids are known inhibitors of serine proteases, the synthesis and binding of a boronic acid analog of the pentapeptide cleavage product, AVKAA, was studied using MycP₁ variants from Mycobacterium thermoresistible (MycP_1mth), Mycobacterium smegmatis (MycP_1msm) and M. tuberculosis (MycP_1mtu). We synthesized the boropentapeptide, HAlaValLysAlaAlaB(OH)₂ (1) and the analogous pinanediol PD-protected HAlaValLysAlaAlaBO₂(PD) (2) using an Fmoc/Boc peptide strategy. The pinanediol boropentapeptide 2 displayed IC₅₀ values 121.6 ± 25.3 μM for MycP_1mth, 93.2 ± 37.3 μM for MycP_1msm and 37.9 ± 5.2 μM for MycP_1mtu. Such relatively strong binding creates a chance for crystalizing the complex with 2 and finding the structure of the unknown MycP₁ catalytic site that would potentially facilitate the development of new anti-tuberculosis drugs.     

Semi-automated program for analysis of local Ca2+ spark release with application for classification of heart cell type

Local Ca²⁺ spark releases are essential to the Ca²⁺ cycling process. Thus, they play an important role in ventricular and atrial cell contraction, as well as in sinoatrial cell automaticity. Characterizing their properties in healthy cells from different regions in the heart can reveal the basic biophysical differences among these regions. We designed a semi-automatic Matlab Graphical User Interface (called Sparkalyzer) to characterize parameters of Ca²⁺ spark release from any major cardiac tissue, as recorded in line-scan mode with a confocal laser-scanning microscope. We validated the algorithm on experimental images from rabbit sinoatrial, atrial, and ventricular cells loaded with Fluo-4 AM. The program characterizes general image parameters of Ca²⁺ transients and sparks: spark duration, which indicates for how long the spark provides Ca²⁺ to the closed intracellular mechanisms (typical value: 25 ± 1, 23 ± 1, 26 ± 1 ms for sinoatrial, atrial, and ventricular cells, respectively); spark amplitude, which indicates the amount of Ca²⁺ released by a single spark (1.6 ± 0.1, 1.6 ± 0.2, 1.4 ± 0.1 F/F₀ for sinoatrial, atrial, and ventricular cells, respectively); spark length, which is the length of the Ca²⁺ wavelets fired out of a row of ryanodine receptors (5 ± 0.1, 5 ± 0.2, 3.4 ± 0.3 μm for sinoatrial, atrial, or ventricular cells, respectively) and number of sparks (0.14 ± 0.02, 0.025 ± 0.01, 0.02 ± 0.01 for 1 μm in 1 s for sinoatrial, atrial, and ventricular cells, respectively). This method is reliable for Ca²⁺ spark analysis of sinoatrial, atrial, or ventricular cells. Moreover, by examining the average value of Ca²⁺ spark characteristics and their scattering around the mean, atrial, ventricular and sinoatrial cells can be differentiated.     

Cleavage furrow organization requires PIP(2)-mediated recruitment of anillin

Cell division is achieved by a plasma membrane furrow that must ingress between the segregating chromosomes during anaphase [1-3]. The force that drives furrow ingression is generated by the actomyosin cytoskeleton, which is linked to the membrane by an as yet undefined molecular mechanism. A key component of the membrane furrow is anillin. Upon targeting to the furrow through its pleckstrin homology (PH) domain, anillin acts as a scaffold linking the actomyosin and septin cytoskeletons to maintain furrow stability (reviewed in [4, 5]). We report that the PH domain of anillin interacts with phosphatidylinositol phosphate lipids (PIPs), including PI(4,5)P(2), which is enriched in the furrow. Reduction of cellular PI(4,5)P(2) or mutations in the PH domain of anillin that specifically disrupt the interaction with PI(4,5)P(2), interfere with the localization of anillin to the furrow. Reduced expression of anillin disrupts symmetric furrow ingression that can be restored by targeting ectopically expressed anillin to the furrow using an alternate PI(4,5)P(2) binding module, a condition where the septin cytoskeleton is not recruited to the plasma membrane. These data demonstrate that the anillin PH domain has two functions: targeting anillin to the furrow by binding to PI(4,5)P(2) to maintain furrow organization and recruiting septins to the furrow.     

Stabilization of anaphase midzone microtubules is regulated by Rho during cytokinesis in human fibrosarcoma cells

The dynamics of astral and midzone microtubules (MTs) must be separately regulated during cell division, but the mechanism of selective stabilization of midzone MTs is poorly understood. Here we show that, in HT1080 cells, activation of Rho is required to stabilize midzone MTs, and to maintain the midzone structures after anaphase onset or during cytokinesis. Ect2-depleted cells undergoing conventional cytokinesis (cytokinesis A) or contractile ring-independent cytokinesis (cytokinesis B) formed abnormally thin bundles of midzone MTs. C3-loaded mitotic cells with inactivated Rho showed similar but more severe disorganization of midzone MTs. In addition, the bundles of astral MTs were abnormally abundant along the cell periphery in both Ect2-depleted and C3-loaded mitotic cells. Mitotic kinesin-like protein 1 (MKLP1), a component of the spindle midzone required for bundling of MTs, was localized only in the narrower equatorial regions in Ect2-depleted cells, and disappeared from the midzone accompanying the progression of the mitotic phase in C3-loaded cells. Stabilization of MTs by taxol was sufficient to maintain the midzone structures in C3-loaded mitotic cells. These results, when combined with a preceding analysis on another, microtubule-associated Rho GEF (C.J. Bakal, D. Finan, J. LaRose, C.D. Wells, G. Gish, S. Kulkarni, P. DeSepulveda, A. Wilde, R. Rottapel, The Rho GTP exchange factor Lfc promotes spindle assembly in early mitosis, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 9529–9534), suggest that mammalian cells have two potential steps that require active Rho for the stabilization of midzone MTs during mitosis and cytokinesis.     

Localization of Pavarotti-KLP in living Drosophila embryos suggests roles in reorganizing the cortical cytoskeleton during the mitotic cycle

Pav-KLP is the Drosophila member of the MKLP1 family essential for cytokinesis. In the syncytial blastoderm embryo, GFP-Pav-KLP cyclically associates with astral, spindle, and midzone microtubules and also to actomyosin pseudocleavage furrows. As the embryo cellularizes, GFP-Pav-KLP also localizes to the leading edge of the furrows that form cells. In mononucleate cells, nuclear localization of GFP-Pav-KLP is mediated through NLS elements in its C-terminal domain. Mutants in these elements that delocalize Pav-KLP to the cytoplasm in interphase do not affect cell division. In mitotic cells, one population of wild-type GFP-Pav-KLP associates with the spindle and concentrates in the midzone at anaphase B. A second is at the cell cortex on mitotic entry and later concentrates in the region of the cleavage furrow. An ATP binding mutant does not localize to the cortex and spindle midzone but accumulates on spindle pole microtubules to which actin is recruited. This leads either to failure of the cleavage furrow to form or later defects in which daughter cells remain connected by a microtubule bridge. Together, this suggests Pav-KLP transports elements of the actomyosin cytoskeleton to plus ends of astral microtubules in the equatorial region of the cell to permit cleavage ring formation.     

Synthesis of triazole Schiff bases: Novel inhibitors of nucleotide pyrophosphatase/phosphodiesterase-1

A series of Schiff base triazoles 1–25 was synthesized and evaluated for their nucleotide pyrophosphatase/phosphodiesterase-1 inhibitory activities. Among twenty-five compounds, three compounds 10 (IC₅₀ = 132.20 ± 2.89 μM), 13 (IC₅₀ = 152.83 ± 2.39 μM), and 22 (IC₅₀ = 251.0 ± 6.64 μM) were identified as potent inhibitors with superior activities than the standard EDTA (IC₅₀ = 277.69 ± 2.52 μM). The newly identified inhibitors may open a new avenue for the development of treatment of phosphodiesterase-I related disorders. These compounds were also evaluated for carbonic anhydrase, acetylcholinesterase and butyrylcholinesterase inhibitory potential and were found to be inactive. The compounds showed non-toxic effect towards PC3 cell lines.     

Abundance and biomass of planktonic ciliates in the sea area around Zhangzi Island,Northern Yellow Sea

We investigated the abundance and biomass of planktonic ciliates in the sea area around Zhangzi Island, Northern Yellow Sea, from July 2009 to June 2010. Ciliates were sampled monthly from surface to bottom with a 10 m depth interval at 13 sample stations along three transects. A 1 L sample of water from each depth was collected with a 2.5 L Niskin water sampler and fixed in 1% acid Lugol’s iodine solution. Water samples were pre-concentrated using the Utermöhl method and observed using an Olympus IX51 inverted microscope at 100× or 200x. The dimensions of the ciliates were measured and the cell volume of each species was estimated using appropriate geometric shapes. The carbon:volume ratio used to calculate biomass was 0.19 pg C/μm³. Abundance and biomass of the ciliate in water column were calculated as the integral of the abundance and biomass from bottom to surface, respectively. The classification of tintinnids was based on taxonomic literature. The average abundance of non-loricate ciliates was 3066 ± 2805 ind/L, ranging from 165 ind/L (50 m depth of St. B6 in July) to 26,595 ind/L (surface of St. C1 in September). The average biomass of non-loricate ciliates was 2.88 ± 2.68 μg C/L, ranging from 0.05 μg C/L (10 m depth of St. A6 in July) to 20.51 μg C/L (surface of St. A5 in August). The average tintinnid abundance was 142 ± 273 ind/L, ranging from 0 ind/L (monthly) to 2756 ind/L (surface of St. A1 in July). The average tintinnid biomass was 0.84 ± 2.19 μg C/L, ranging from 0.00 μg C/L (every month) to 37.64 μg C/L (20 m depth of St. C5 in July). The results showed that the average abundance of total ciliates was 3208 ± 2828 ind/L, ranging from 166 ind/L (10 m depth of St. A6 in July) to 26,625 ind/L (surface of St. C1 in September); the average biomass of total ciliates was 3.73 ± 3.55 μg C/L, ranging from 0.05 μg C/L (10 m depth of St. A6 in July) to 38.29 μg C/L (20 m depth of St. C5 in July). Abundance and biomass were vertically homogeneous in February, November and December, but decreased dramatically from the surface down to the bottom in other months. 23 tintinnid species were identified, 12 of which were in genus Tintinnopsis. Tintinnid species were more abundant in February, July and August. Tintinnids occupied 6.6 ± 10.2% and 19.7 ± 23.3% of the total ciliate abundance and biomass, respectively, which increased during the warm season and at coastal stations, and decreased during the cold season and at offshore stations. Large non-loricate ciliate species were prevalent in spring, while smaller species dominated in summer and autumn. The average abundance of total ciliates in water column was 132 ± 72 × 10⁶ ind/m², with increases during spring and autumn. The average biomass of total ciliates in water column was 152.57 ± 93.10 mg C/m², with increases during spring and summer. The average abundance and biomass of total ciliates in water column were greater at offshore stations than at coastal stations during spring and autumn, and were lower during summer and winter. Non-loricate ciliates, tintinnids and total ciliates showed significant positive correlation with temperature and significant negative correlation with salinity (p < 0.01). Non-loricate ciliates and total ciliates showed significant positive correlation with Chl a concentration (p < 0.01); however, relationship between Chl a concentration and tintinnids was not significant.     

Morphology and phylogeny of Henneguya jocu n. sp. (Myxosporea,Myxobolidae), infecting the gills of the marine fish Lutjanus jocu

Henneguya jocu n. sp. (Myxosporea, Myxobolidae) is described from the gill lamellae of the marine teleost fish Lutjanus jocu, with a focus on ultrastructural and molecular features. This myxosporean forms subspherical cysts up to ∼260 μm × 130 μm long, and develops asynchronously. Mature myxospores ellipsoidal with a bifurcated caudal process. Myxospore length 10.9 ± 0.4 μm (n = 50); width, 8.2 ± 0.3 μm (n = 50); and thickness, 2.9 ± 0.5 μm (n = 50). Two equal caudal processes, 34.1 ± 1.0 μm long (n = 50); and total myxospore length, 45.2 ± 1.0 μm (n = 50). Two symmetric valves surround two ellipsoidal polar capsules, 5.0 ± 0.3 × 1.4 ± 0.2 μm (n = 20), each containing an isofilar polar filament forming 4–5 coils along the inner wall of these structures, as well as a binucleated sporoplasm presenting a spherical vacuole and several globular sporoplasmosomes. Both the morphological data and molecular analysis of the SSU rDNA gene identify this parasite as a new species of the genus Henneguya. Maximum Likelihood and Maximum Parsimony analyses further indicate that the parasite clusters within others marine Myxobolidae species, forming a group alongside other Henneguya species described from marine hosts.     

Caloxin 1b3: A novel plasma membrane Ca2+-pump isoform 1 selective inhibitor that increases cytosolic Ca2+ in endothelial cells

The purpose of this study was to invent an extracellular inhibitor selective for the plasma membrane Ca²⁺ pump(s) (PMCA) isoform 1. PMCA extrude Ca²⁺ from cells during signalling and homeostasis. PMCA isoforms are encoded by 4 genes (PMCA1–4). Pig coronary artery endothelium and smooth muscle express the genes PMCA1 and 4. We showed that the endothelial cells contained mostly PMCA1 protein while smooth muscle cells had mostly PMCA4. A random peptide phage display library was screened for binding to synthetic extracellular domain 1 of PMCA1. The selected phage population was screened further by affinity chromatography using PMCA from rabbit duodenal mucosa which expressed mostly PMCA1. The peptide displayed by the selected phage was termed caloxin 1b3. Caloxin 1b3 inhibited PMCA Ca²⁺–Mg²⁺-ATPase in the rabbit duodenal mucosa (PMCA1) with a greater affinity (inhibition constant = 17 ± 2 μM) than the PMCA in the human erythrocyte ghosts (PMCA4, inhibition constant = 45 ± 4 μM). The affinity of caloxin 1b3 was also higher for PMCA1 than for PMCA2 and 3 indicating its selectivity for PMCA1. Consistent with an inhibition of PMCA1, caloxin 1b3 addition to the medium increased cytosolic Ca²⁺ concentration in endothelial cells. Caloxin 1b3 is the first known PMCA1 selective inhibitor. We anticipate caloxin 1b3 to aid in understanding PMCA physiology in endothelium and other tissues.     

Ciliogenesis in cryopreserved mammalian tracheal epithelial cells cultured at the air–liquid interface

To determine air–liquid interface (ALI) culture derived from cryopreserved mammalian tracheal ciliated cells is a viable ciliated cell model for the investigations of regulatory mechanisms of ciliary beat frequency (CBF), two studies were performed using ovine and porcine tracheae obtained from local slaughterhouses. The protease-digested tracheal ciliated cells were harvested and cultured at the ALI using collagen-coated, porous membrane inserts. In study 1, the ALI culturing protocols were established using non-cryopreserved ovine tracheal ciliated cells. Ciliogenesis was documented with immuno-histology and electron micrographs. Vigorous beating cilia were video-recorded. CBF was measured by laser light scattering. The functional integrity of the autonomic receptors of the ciliated cells was confirmed with the stimulatory responses of CBF using luminal methacholine and basolateral terbutaline. In study 2, porcine tracheal ciliated cells stored in liquid nitrogen for a minimum of 4 weeks were used. The cryopreserved cells were thawed and cultured using the ALI protocol established in study 1. After two months, cilia outgrowths were confirmed using video microscopy and scanning electron micrograph (SEM). The trans-epithelial resistances were 28.5 kΩ (n = 4). Luminal applications of 1 μM and 10 μM methacholine stimulated CBF from a baseline of 7.4 ± 0.2 Hz to 8.4 ± 0.8 Hz and 7.7 ± 0.4 Hz, respectively (n = 5). Basolateral applications of 1 μM and 10 μM terbutaline stimulated CBF from a baseline of 7.5 ± 0.3 Hz to 8.2 ± 0.4 Hz and 8.0 ± 0.4 Hz, respectively (n = 5). These data demonstrated that a ciliated cell bank can be established using cryopreserved ciliated cells for pulmonary drug discovery and toxicological screening.     

Interaction between EB1 and p150glued is required for anaphase astral microtubule elongation and stimulation of cytokinesis

In animal cells, microtubules (MTs) of the mitotic apparatus (MA) communicate with the cell cortex to stimulate cytokinesis; however, the molecular nature of this stimulus remains elusive . A signal for cytokinesis likely involves the MT plus end binding family of proteins, which includes EB1, p150glued, APC, LIS1, and CLIP-170. These proteins modulate MT dynamics and facilitate interactions between growing MTs and their intracellular targets, including kinetochores, organelles, and the cell cortex . The dynein-dynactin complex mediates many of these microtubule capture events . We report that EB1 and p150glued interactions are required for stimulation of cytokinesis in dividing sea urchin eggs. Injected antibodies against EB1 or p150glued suppressed furrow ingression but did not prevent elongation of anaphase astral MTs toward the cortex, suggesting that EB1 and dynactin are both required for communication between the MA and the cortex. Targeted disruption of the interaction between EB1 and p150glued suppressed anaphase astral MT elongation and resulted in a delay of cytokinesis that could not be overcome by manipulation of the asters toward the cortex. We conclude that EB1 and dynactin participate in stimulation of the cleavage furrow, and their interaction promotes elongation of astral MTs at anaphase onset.