Live-cell imaging and ultrastructural analysis reveal remarkable features of cultured porcine gonocytes.

Gonocytes in the neonatal testis have male germline stem cell potential. The objective of the present study was to examine the behavior and ultrastructure of gonocytes in culture. Neonatal porcine testis cells were cultured for 4 weeks and underwent live-cell imaging to explore real-time interactions among cultured cells. This included imaging every 1 h from day 0 to day 3, every 2 h from day 4 to day 7, and every 1 h for 24 h at days 14, 21, and 28. Samples also underwent scanning electron microscopy, transmission electron microscopy, morphometric evaluations, immunofluorescence, and RT-PCR. Live-cell imaging revealed an active amoeboid-like movement of gonocytes, assisted by the formation of extensive cytoplasmic projections, which, using scanning electron microscopy, were categorized into spike-like filopodia, leaf-like lamellipodia, membrane ruffles, and cytoplasmic blebs. In the first week of culture, gonocytes formed loose attachments on top of a somatic cell monolayer and, in week 2, formed grape-like clusters, which, over time, grew in cell number. Starting at week 3 of culture, some of the gonocyte clusters transformed into large multinucleated embryoid body-like colonies (EBLCs) that expressed both gonocyte- and pluripotent-specific markers. The number and diameter of individual gonocytes, the number and density of organelles within gonocytes, as well as the number and diameter of the EBLCs increased over time (P < 0.05). In conclusion, cultured porcine gonocytes displayed extensive migratory behavior facilitated by their various cytoplasmic projections, propagated, and transformed into EBLCs that increased in size and complexity over time.

Homing and Engraftment of Intravenously Administered Equine Cord Blood-Derived Multipotent Mesenchymal Stromal Cells to Surgically Created Cutaneous Wound in Horses: A Pilot Project.

Limb wounds on horses are often slow to heal and are prone to developing exuberant granulation tissue (EGT) and close primarily through epithelialization, which results in a cosmetically inferior and non-durable repair. In contrast, wounds on the body heal rapidly and primarily through contraction and rarely develop EGT. Intravenous (IV) multipotent mesenchymal stromal cells (MSCs) are promising. They home and engraft to cutaneous wounds and promote healing in laboratory animals, but this has not been demonstrated in horses. Furthermore, the clinical safety of administering >1.00 × 108 allogeneic MSCs IV to a horse has not been determined. A proof-of-principle pilot project was performed with two horses that were administered 1.02 × 108 fluorescently labeled allogeneic cord blood-derived MSCs (CB-MSCs) following wound creation on the forelimb and thorax. Wounds and contralateral non-wounded skin were sequentially biopsied on days 0, 1, 2, 7, 14, and 33 and evaluated with confocal microscopy to determine presence of homing and engraftment. Results confirmed preferential homing and engraftment to wounds with persistence of CB-MSCs at 33 days following wound creation, without clinically adverse reactions to the infusion. The absence of overt adverse reactions allows further studies to determine effects of IV CB-MSCs on equine wound healing.

Fermitin family homolog-2 (FERMT2) is highly expressed in human placental villi and modulates trophoblast invasion.

BACKGROUND: Integrins are transmembrane receptors that mediate cell-extracellular matrix (ECM) and cell-cell adhesion and trophoblast cells undergo changes in integrin expression as they differentiate. However, the mechanism(s) of integrin activation leading to integrin-mediated signaling in trophoblast cell differentiation is unknown. The Fermitin family proteins are integrin activators that help mediate integrin-mediated signaling, but have never been studied in detail within the human placenta. Thus, we examined the spatiotemporal pattern of expression of Fermitin family homolog-2 (FERMT2) in human chorionic villi throughout gestation and its role in trophoblast-substrate adhesion and invasion. METHODS: Placental villous tissue was obtained from patients undergoing elective terminations by dilatation and curettage at weeks 8-12 (n = 10), weeks 13-14 (n = 8), as well as from term deliveries at weeks 37-40 (n = 6). Tissues were fixed, processed and sections utilized for immunofluorescence analysis of FERMT2 expression during gestation. Additionally, HTR8-SVneo human trophoblast cells were transfected by electroporation with FERMT2-specific siRNAs or non-targeting siRNAs (control) and used in cell-substrate adhesion as well as invasion assays. RESULTS: FERMT2 was more commonly expressed in the basal domain of villous cytotrophoblast cells and prominently localized around the periphery of individual extravillous trophoblast cells. siRNA-mediated knockdown of FERMT2 in HTR8-SVneo cells resulted in significantly decreased trophoblast-substrate attachment (p < 0.05) as well as significantly decreased trophoblast invasion (p < 0.05) relative to control cells. CONCLUSIONS: The detection of FERMT2 throughout extravillous trophoblast columns and the results of invasion assays demonstrated that this protein is likely an important regulator of integrin activation in extravillous cells to modulate migration and invasion.

Stat3 regulates centrosome clustering in cancer cells via Stathmin/PLK1.

Cancer cells frequently have amplified centrosomes that must be clustered together to form a bipolar mitotic spindle, and targeting centrosome clustering is considered a promising therapeutic strategy. A high-content chemical screen for inhibitors of centrosome clustering identified Stattic, a Stat3 inhibitor. Stat3 depletion and inhibition in cancer cell lines and in tumours in vivo caused significant inhibition of centrosome clustering and viability. Here we describe a transcription-independent mechanism for Stat3-mediated centrosome clustering that involves Stathmin, a Stat3 interactor involved in microtubule depolymerization, and the mitotic kinase PLK1. Furthermore, PLK4-driven centrosome amplified breast tumour cells are highly sensitive to Stat3 inhibitors. We have identified an unexpected role of Stat3 in the regulation of centrosome clustering, and this role of Stat3 may be critical in identifying tumours that are sensitive to Stat3 inhibitors.

Microtubules in Plant Cells: Strategies and Methods for Immunofluorescence, Transmission Electron Microscopy, and Live Cell Imaging.

Microtubules (MTs) are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labeling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high-pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography.

Identification of novel small molecule inhibitors of centrosome clustering in cancer cells.

Most normal cells have two centrosomes that form bipolar spindles during mitosis, while cancer cells often contain more than two, or "supernumerary" centrosomes. Such cancer cells achieve bipolar division by clustering their centrosomes into two functional poles, and inhibiting this process then leads to cancer-specific cell death. A major problem with clinically used anti-mitotic drugs, such as paclitaxel, is their toxicity in normal cells. To discover new compounds with greater specificity for cancer cells, we established a high-content screen for agents that block centrosome clustering in BT-549 cells, a breast cancer cell line that harbors supernumerary centrosomes. Using this screen, we identified 14 compounds that inhibit centrosome clustering and induce mitotic arrest. Some of these compounds were structurally similar, suggesting a common structural motif important for preventing centrosome clustering. We next compared the effects of these compounds on the growth of several breast and other cancer cell lines, an immortalized normal human mammary epithelial cell line, and progenitor-enriched primary normal human mammary epithelial cells. From these comparisons, we found some compounds that kill breast cancer cells, but not their normal epithelial counterparts, suggesting their potential for targeted therapy. One of these compounds, N2-(3-pyridylmethyl)-5-nitro-2-furamide (Centrosome Clustering Chemical Inhibitor-01, CCCI-01), that showed the greatest differential response in this screen was confirmed to have selective effects on cancer as compared to normal breast progenitors using more precise apoptosis induction and clonogenic growth endpoints. The concentration of CCCI-01 that killed cancer cells in the clonogenic assay spared normal human bone marrow hematopoietic progenitors in the colony-forming cell assay, indicating a potential therapeutic window for CCCI-01, whose selectivity might be further improved by optimizing the compound. Immunofluorescence analysis showed that treatment with CCCI-01 lead to multipolar spindles in BT-549, while maintaining bipolar spindles in the normal primary human mammary epithelial cells. Since centrosome clustering is a complex process involving multiple pathways, the 14 compounds identified in this study provide a potentially novel means to developing non-cross-resistant anti-cancer drugs that block centrosome clustering.

Integrin-linked kinase regulates interphase and mitotic microtubule dynamics.

Integrin-linked kinase (ILK) localizes to both focal adhesions and centrosomes in distinct multiprotein complexes. Its dual function as a kinase and scaffolding protein has been well characterized at focal adhesions, where it regulates integrin-mediated cell adhesion, spreading, migration and signaling. At the centrosomes, ILK regulates mitotic spindle organization and centrosome clustering. Our previous study showed various spindle defects after ILK knockdown or inhibition that suggested alteration in microtubule dynamics. Since ILK expression is frequently elevated in many cancer types, we investigated the effects of ILK overexpression on microtubule dynamics. We show here that overexpressing ILK in HeLa cells was associated with a shorter duration of mitosis and decreased sensitivity to paclitaxel, a chemotherapeutic agent that suppresses microtubule dynamics. Measurement of interphase microtubule dynamics revealed that ILK overexpression favored microtubule depolymerization, suggesting that microtubule destabilization could be the mechanism behind the decreased sensitivity to paclitaxel, which is known to stabilize microtubules. Conversely, the use of a small molecule inhibitor selective against ILK, QLT-0267, resulted in suppressed microtubule dynamics, demonstrating a new mechanism of action for this compound. We further show that treatment of HeLa cells with QLT-0267 resulted in higher inter-centromere tension in aligned chromosomes during mitosis, slower microtubule regrowth after cold depolymerization and the presence of a more stable population of spindle microtubules. These results demonstrate that ILK regulates microtubule dynamics in both interphase and mitotic cells.

The N-terminal TOG domain of Arabidopsis MOR1 modulates affinity for microtubule polymers.

Microtubule-associated proteins of the highly conserved XMAP215/Dis1 family promote both microtubule growth and shrinkage, and move with the dynamic microtubule ends. The plant homologue, MOR1, is predicted to form a long linear molecule with five N-terminal TOG domains. Within the first (TOG1) domain, the mor1-1 leucine to phenylalanine (L174F) substitution causes temperature-dependent disorganization of microtubule arrays and reduces microtubule growth and shrinkage rates. By expressing the two N-terminal TOG domains (TOG12) of MOR1, both in planta for analysis in living cells and in bacteria for in vitro microtubule-binding and polymerization assays, we determined that the N-terminal domain of MOR1 is crucial for microtubule polymer binding. Tagging TOG12 at the N-terminus interfered with its ability to bind microtubules when stably expressed in Arabidopsis or when transiently overexpressed in leek epidermal cells, and impeded polymerase activity in vitro. In contrast, TOG12 tagged at the C-terminus interacted with microtubules in vivo, rescued the temperature-sensitive mor1-1 phenotype, and promoted microtubule polymerization in vitro. TOG12 constructs containing the L174F mor1-1 point mutation caused microtubule disruption when transiently overexpressed in leek epidermis and increased the affinity of TOG12 for microtubules in vitro. This suggests that the mor1-1 mutant protein makes microtubules less dynamic by binding the microtubule lattice too strongly to support rapid plus-end tracking. We conclude from our results that a balanced microtubule affinity in the N-terminal TOG domain is crucial for the polymerase activity of MOR1.

Mechanisms of leaf tooth formation in Arabidopsis.

Serration found along leaf margins shows species-specific characters. Whereas compound leaf development is well studied, the process of serration formation is largely unknown. To understand mechanisms of serration development, we investigated distinctive features of cells that could give rise to tooth protrusion in the simple-leaf plant Arabidopsis. After the emergence of a tooth, marginal cells, except for cells at the sinuses and tips, started to elongate rapidly. Localized cell division seemed to keep cells at the sinus smaller, rather than halt cell elongation. As leaves matured, the marginal cell number between teeth became similar in any given tooth. These results suggest that teeth are formed by repetition of an unknown mechanism that spatially monitors cell number and regulates cell division. We then examined the role of CUP-SHAPED COTYLEDON 2 (CUC2) in serration development. cuc2-3 forms fewer hydathodes and auxin maxima, visualized by DR5rev::GFP, at the leaf margin, suggesting that CUC2 patterns serration through the regulation of auxin. In contrast to a previous interpretation, comparison of leaf outlines revealed that CUC2 promotes outgrowth of teeth rather than suppression of growth at the sinuses. We found that mutants with increased CUC2 expression form ectopic tissues and mis-express SHOOT MERISTEMLESS (STM) at the sinus between the enhanced teeth. Similar but infrequent STM expression was found in the wild type, indicating STM involvement in the serration of simple leaves. Our study provides insights into the morphological and molecular mechanisms for leaf development and tooth formation, and highlights similarities between serration and compound leaf development.

Strategies for imaging microtubules in plant cells.

Microtubules are required throughout plant development for a wide variety of processes, and different strategies have been evolved to visualize them. This chapter summarizes the most effective of these methods and points out potential problems and pitfalls. We outline the freeze-shattering method for immunolabeling microtubules in aerial organs such as leaves that require mechanical permeabilization, discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide different fixation protocols for preserving MTs for transmission electron microscopy including chemical fixation, high pressure freezing/freeze substitution, and post-fixation staining procedures for transmission electron microscopy.

Amphotericin B-induced renal tubular cell injury is mediated by Na+ Influx through ion-permeable pores and subsequent activation of mitogen-activated protein kinases and elevation of intracellular Ca2+ concentration.

Amphotericin B (AMB) is one of the most effective antifungal agents; however, its use is often limited by the occurrence of adverse events, especially nephrotoxicity. The present study was designed to determine the possible mechanisms underlying the nephrotoxic action of AMB. The exposure of a porcine proximal renal tubular cell line (LLC-PK1 cells) to AMB caused cell injury, as assessed by mitochondrial enzyme activity, the leakage of lactate dehydrogenase, and tissue ATP depletion. Propidium iodide uptake was enhanced, while terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was not affected by AMB, suggesting a lack of involvement of apoptosis in AMB-induced cell injury. The cell injury was inhibited by the depletion of membrane cholesterol with methyl-beta-cyclodextrin, which lowered the extracellular Na(+) concentration or the chelation of intracellular Ca(2+). The rise in the intracellular Ca(2+) concentration may be mediated through the activation of the ryanodine receptor (RyR) on the endoplasmic reticulum and the mitochondrial Na(+)-Ca(2+) exchanger, since cell injury was attenuated by dantrolene (an RyR antagonist) and CGP37157 (an Na(+)-Ca(2+) exchanger inhibitor). Moreover, AMB-induced cell injury was reversed by PD169316 (a p38 mitogen-activated protein [MAP] kinase inhibitor), c-Jun N-terminal kinase inhibitor II, and PD98059 (a MEK1/2 inhibitor). The phosphorylations of these MAP kinases were enhanced by AMB in a calcium-independent manner, suggesting the involvement of MAP kinases in AMB-induced cell injury. These findings suggest that Na(+) entry through membrane pores formed by the association of AMB with membrane cholesterol leads to the activation of MAP kinases and the elevation of the intracellular Ca(2+) concentration, leading to renal tubular cell injury.

MOR1, the Arabidopsis thaliana homologue of Xenopus MAP215, promotes rapid growth and shrinkage, and suppresses the pausing of microtubules in vivo.

MOR1, the Arabidopsis thaliana homologue of the Xenopus microtubule-associated protein MAP215, is required for spatial organization of the acentrosomal microtubule arrays of plant cells. To determine how loss of MOR1 function affects microtubule dynamics, we compared various parameters of microtubule dynamics in the temperature-sensitive mor1-1 mutant at its permissive and restrictive temperatures, 21 degrees C and 31 degrees C, respectively. Dynamic events were tracked in live cells expressing either GFP-tagged beta-tubulin or the plus end tracking EB1. Microtubule growth and shrinkage velocities were both dramatically reduced in mor1-1 at 31 degrees C and the incidence and duration of pause events increased. Interestingly, the association of EB1 with microtubule plus ends was reduced in mor1-1 whereas side wall binding increased, suggesting that MOR1 influences the association of EB1 with microtubules either by modulating microtubule plus end structure or by interacting with EB1. Although mor1-1 microtubules grew and shrank more slowly than wild-type microtubules at 21 degrees C, the incidence of pause was not altered, suggesting that pause events, which occur more frequently at 31 degrees C, have a major detrimental role in the spatial organization of cortical microtubules. Extensive increases in microtubule dynamics in wild-type cells when shifted from 21 degrees C to 31 degrees C underline the importance of careful temperature control in live cell imaging.

MICROTUBULE ORGANIZATION 1 regulates structure and function of microtubule arrays during mitosis and cytokinesis in the Arabidopsis root.

MICROTUBULE ORGANIZATION 1 (MOR1) is a plant member of the highly conserved MAP215/Dis1 family of microtubule-associated proteins. Prior studies with the temperature-sensitive mor1 mutants of Arabidopsis (Arabidopsis thaliana), which harbor single amino acid substitutions in an N-terminal HEAT repeat, proved that MOR1 regulates cortical microtubule organization and function. Here we demonstrate by use of live cell imaging and immunolabeling that the mor1-1 mutation generates specific defects in the microtubule arrays of dividing vegetative cells. Unlike the universal cortical microtubule disorganization in elongating mor1-1 cells, disruption of mitotic and cytokinetic microtubule arrays was not detected in all dividing cells. Nevertheless, quantitative analysis identified distinct defects in preprophase bands (PPBs), spindles, and phragmoplasts. In nearly one-half of dividing cells at the restrictive temperature of 30 degrees C, PPBs were not detected prior to spindle formation, and those that did form were often disrupted. mor1-1 spindles and phragmoplasts were short and abnormally organized and persisted for longer times than in wild-type cells. The reduced length of these arrays predicts that the component microtubule lengths are also reduced, suggesting that microtubule length is a critical determinant of spindle and phragmoplast structure, orientation, and function. Microtubule organizational defects led to aberrant chromosomal arrangements, misaligned or incomplete cell plates, and multinucleate cells. Antiserum raised against an N-terminal MOR1 sequence labeled the full length of microtubules in interphase arrays, PPBs, spindles, and phragmoplasts. Continued immunolabeling of the disorganized and short microtubules of mor1-1 at the restrictive temperature demonstrated that the mutant mor1-1(L174F) protein loses function without dissociating from microtubules, providing important insight into the mechanism by which MOR1 may regulate microtubule length.