The obligate root parasite Cistanche armena is a recently rediscovered, extremely rare endangered species endemic to Armenia, specifically parasitizing camelthorn (Alhagi maurorum, Fabaceae) and saltwort (Salsola dendroides, Chenopodiaceae). Its populations are reputedly declining due to habitat destruction and biotic impacts. Since the only known means of its reproduction is via the seeds, understanding the mechanisms of breaking C. armena seed dormancy and germination along with the related aspects of the species’ biology is highly important both from fundamental (functional ecology and evolution) and applied (conservation and management) perspectives. Here, we present the first in vitro seed germination protocol for C. armena involving fluridone, a systemic herbicide targeting the carotenoid biosynthetic pathway. In addition, the seed micromorphology of C. armena is described using both light microscopy and lignin autofluorescence visualized by confocal laser scanning microscopy. The actin cytoskeleton in radicle cells of germinated C. armena seedlings is described for the first time, being the proof of seed viability. Further elaboration and application of the proposed germination protocol with the cultivation of C. armena on susceptible hosts are altogether seen as a valuable tool for the conservation of this species.

Cell division is a tightly regulated mechanism, notably in tissues where malfunctions can lead to tumour formation or developmental defects. This is particularly true in land plants, where cells cannot relocate and therefore cytokinesis determines tissue topology. In plants, cell division is executed in radically different manners than in animals, with the appearance of new structures and the disappearance of ancestral mechanisms. Whilst F-actin and microtubules closely co-exist, recent studies mainly focused on the involvement of microtubules in this key process. Here, we used a root tracking system to image the spatio-temporal dynamics of both F-actin reporters and cell division markers in dividing cells embedded in their tissues. In addition to the F-actin accumulation at the phragmoplast, we observed and quantified a dynamic apico-basal enrichment of F-actin from the prophase/metaphase transition until the end of the cytokinesis.

Studies on the mechanics of plant cells usually focus on understanding the effects of turgor pressure and properties of the cell wall (CW). While the functional roles of the underlying cytoskeleton have been studied, the extent to which it contributes to the mechanical properties of cells is not elucidated. Here, we study the contributions of the CW, microtubules (MTs) and actin filaments (AFs), in the mechanical properties of Nicotiana tabacum cells. We use a multiscale biomechanical assay comprised of atomic force microscopy and micro-indentation in solutions that (i) remove MTs and AFs and (ii) alter osmotic pressures in the cells. To compare measurements obtained by the two mechanical tests, we develop two generative statistical models to describe the cell’s behaviour using one or both datasets. Our results illustrate that MTs and AFs contribute significantly to cell stiffness and dissipated energy, while confirming the dominant role of turgor pressure.

Prolyl endopeptidase (PREP) is a post-proline cleaving enzyme. It is involved in the regulation of multiple inositol polyphosphate phosphatase activity implicated in the pathway of inositol 1,4,5-trisphosphate, resulting in the modulation of cytosolic Ca2+ levels. Besides its peptidase activity, PREP was identified as a binding partner of tubulin, suggesting that it may participate in microtubule-associate processes. In this paper, we evaluated the expression of PREP mRNA and protein by polymerase chain reaction and western blot analyses and its co-localization with tubulin by immunofluorescence in adult mouse seminal vesicles. We showed that both proteins are cytoplasmic: tubulin is localized at the apical half part of the cell, while PREP has a more diffuse localization, showing a prominent distribution at the apical cytoplasm. These findings support our hypothesis of a specific role for PREP in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular its association with tubulin filaments. Moreover, it may regulate Ca2+ levels, and promote the final step of vesicular exocytosis, namely the fusion of the vesicles with the plasma membrane. These results strongly suggest that there is a pivotal role for PREP in vesicle exocytosis, as well as in the physiology of mouse seminal vesicles.

Digyny, the presence of a third pronucleus due to the failure of second polar body extrusion, is problematic after intracytoplasmic sperm injection (ICSI) practices. Mitochondria have critical roles such as production of adenosine triphosphate (ATP) and regulation of Ca2+ homeostasis during oocyte maturation, fertilization and the following development, while the regulation of meiotic spindle formation, chromosome segregation, pronuclear apposition and cytokinesis is closely associated with the cytoskeleton. In this study, mitochondrial membrane potential, distribution of F-actin and γ-tubulin, and the ultrastructure of three pronuclear (3PN) oocytes were investigated. 3PN oocytes after ICSI procedure were taken from patients who were enrolled in assisted reproduction programmes. For mitochondrial membrane potential analysis, fresh oocytes stained with the mitochondrial membrane potential probe JC-1, were evaluated under fluorescence microscopy. The mitochondrial membrane potential of three pronuclear oocytes showed similar results to normal zygotes. γ-Tubulin was stained strongly at the subplasmalemmal domain and microfilaments were localized at the cortical, but not the perinuclear, area. Cytoplasmic halos were moderately or not detected by electron microscopy; lipofuscin granules, degenerated mitochondria, and multilamellated bodies were seen in the ooplasm. Immunohistochemistry and electron microscopic findings suggested that mitochondrial membrane potential has no direct effect on second polar body extrusion. This abnormality can be associated with an altered cytoskeleton due to poor oocyte quality.

Confocal microscopy was used to image stages of equine zygote development, at timed intervals, after intracytoplasmic sperm injection (ICSI) of oocytes that were matured in vivo or in vitro. After fixation for 4, 6, 8, 12, or 16 h after ICSI, zygotes were incubated with α/β tubulin antibodies and human anticentromere antibody (CREST/ACA), washed, incubated in secondary antibodies, conjugated to either Alexa 488 or Alexa 647, and incubated with 561-Phalloidin and Hoechst 33258. An Olympus IX81 spinning disk confocal microscope was used for imaging. Data were analyzed using χ2 and Fisher’s exact tests. Minor differences in developmental phases were observed for oocytes matured in vivo or in vitro. Oocytes formed pronuclei earlier when matured in vivo (67% at 6 h and 80% at 8 h) than in vitro (13% at 6 and 8 h); 80% of oocytes matured in vitro formed pronuclei by 12 h. More (p=0.04) zygotes had atypical phenotypes, indicative of a failure of normal zygote development, when oocyte maturation occurred in vitro versus in vivo (30 and 11%, respectively). Some potential zygotes from oocytes matured in vivo had normal phenotypes, although development appeared to be delayed or arrested. Confocal microscopy provided a feasible method to assess equine zygote development using limited samples.

Actin-based cytoskeleton (CSK) and microtubules may bind to RNAs and related molecules implicated in translation. However, many questions remain to be answered regarding the role of cytoskeletal components in supporting the proteins involved in steps in the maturation and translation processes. Here, we performed co-immunoprecipitation and immunofluorescence to examine the association between spectrins, keratins and tubulin and proteins involved in 60S ribosomal maturation and translation in Xenopus stage I oocytes, including ribosomal rpl10, eukaryotic initiation factor 6 (Eif6), thesaurins A/B, homologs of the eEF1α elongation factor, and P0, the ribosomal stalk protein. We found that rpl10 and eif6 cross-reacted with the actin-based CSK and with tubulin. rpl10 co-localizes with spectrin, particularly in the perinuclear region. eif6 is similarly localized. Given that upon ribosomal maturation, the insertion of rpl10 into the 60S subunit occurs simultaneously with the release of eif6, one can hypothesise that actin-based CSK and microtubules provide the necessary scaffold for the insertion/release of these two molecules and, subsequently, for eif6 transport and binding to the mature 60S subunit. P0 and thesaurins cross-reacted with only spectrin and cytokeratins. Thesaurins aggregated at the oocyte periphery, rendering this a territory favourable site for protein synthesis; the CSK may support the interaction between thesaurins and sites of the translating ribosome. Moreover, given that the assembly of the ribosome stalk, where P0 is located, to the 60S subunit is essential for the release of eif6, it can be hypothesised that the CSK can facilitate the binding of the stalk to the 60S.

We investigated the structural complexity and texture of the cytoskeleton and nucleus in human mesenchymal stem cells during early phase differentiation into osteoblasts according to the differentiation–induction method: mechanical and/or chemical stimuli. For this, fractal dimension and a number of parameters utilizing the gray-level co-occurrence matrix (GLCM) were calculated based on single-cell images after confirmation of differentiation by immunofluorescence staining. The F-actin and nuclear fractal dimensions were greater in both stimulus groups compared with the control group. The GLCM values for energy and homogeneity were lower in fibers of the F-actin cytoskeleton, indicating a dispersed F-actin arrangement during differentiation. In the nuclei of both stimulus groups, higher values for energy and homogeneity were calculated, indicating that the chromatin arrangement was chaotic during the early phase of differentiation. It was shown and confirmed that combined stimulation with mechanical and chemical factors accelerated differentiation, even in the early phase. Fractal dimension analysis and GLCM methods have the potential to provide a framework for further investigation of stem cell differentiation.

Free-living amoebae belonging to the genus Acanthamoeba are the causative agents of infections such as amoebic keratitis (AK), granulomatous amoebic encephalitis (GAE) and cutaneous lesions. The mechanisms involved in the establishment of infection are unknown. However, it is accepted that the initial phase of pathogenesis involves adherence to the host tissue. In this work, we analysed surface molecules with an affinity for epithelial and neuronal cells from the trophozoites of Acanthamoeba castellanii. We also investigated the cellular mechanisms that govern the process of trophozoite adhesion to the host cells. We first used confocal and epifluorescence microscopy to examine the distribution of the A. castellanii actin cytoskeleton during interaction with the host cells. The use of drugs, as cytochalasin B (CB) and latrunculin B (LB), revealed the participation of cytoskeletal filaments in the adhesion process. In addition, to identify the proteins and glycoproteins on the surface of A. castellanii, the trophozoites were labelled with biotin and biotinylated lectins. The results revealed bands of surface proteins, some of which were glycoproteins with mannose and N-acetylglucosamine residues. Interaction assays of biotinylated amoebae proteins with epithelial and neuronal cells showed that some surface proteins had affinity for both cell types. The results of this study provide insight into the biochemical and cellular mechanisms of the Acanthamoeba infection process.

Trypanosoma brucei is the etiological agent of devastating parasitic disease in humans and livestock in sub-saharan Africa. The pathogenicity and growth of the parasite are intimately linked to its shape and form. This is in turn derived from a highly ordered microtubule cytoskeleton that forms a tightly arrayed cage directly beneath the pellicular membrane and numerous other cytoskeletal structures such as the flagellum. The parasite undergoes extreme changes in cellular morphology during its life cycle and cell cycles which require a high level of integration and coordination of cytoskeletal processes. In this review we will discuss the role that proteomics techniques have had in advancing our understanding of the molecular composition of the cytoskeleton and its functions. We then consider future opportunities for the application of these techniques in terms of addressing some of the unanswered questions of trypanosome cytoskeletal cell biology with particular focus on the differences in the composition and organisation of the cytoskeleton through the trypanosome life-cycle.

Rod and cone photoreceptors possess ribbon synapses that assist in the transmission of graded light responses to second-order bipolar and horizontal cells of the vertebrate retina. Proper functioning of the synapse requires the juxtaposition of presynaptic release sites immediately adjacent to postsynaptic receptors. In this review, we focus on the synaptic, cytoskeletal, and extracellular matrix proteins that help to organize photoreceptor ribbon synapses in the outer plexiform layer. We examine the proteins that foster the clustering of release proteins, calcium channels, and synaptic vesicles in the presynaptic terminals of photoreceptors adjacent to their postsynaptic contacts. Although many proteins interact with one another in the presynaptic terminal and synaptic cleft, these protein–protein interactions do not create a static and immutable structure. Instead, photoreceptor ribbon synapses are remarkably dynamic, exhibiting structural changes on both rapid and slow time scales.

The role of centrosomes in stem cell division has recently been highlighted and further ascribes important functions to centrosomes in stem cell maintenance, cellular differentiation, and development. Advanced cell and molecular studies coupled with immunofluorescence, electron microscopy, and live cell imaging of specific centrosome proteins have contributed greatly to our knowledge of centrosome composition, structure, and dynamics and have uncovered new insights into mechanisms of centrosome functions in asymmetric cell division. The establishment of asymmetry and differential positioning of mother and daughter centrosomes during stem cell mitosis is important for allowing one cell to maintain stem cell characteristics while the sibling cell undergoes differentiation. Another key role for centrosomes has been revealed in primary cilia of embryonic stem cells that play significant roles in cellular signaling and are therefore critically important for stem cell decisions. Studies of signaling through primary cilia may contribute important information that may aid in the production of specific cells that are suitable for tissue repair and regeneration in the field of regenerative medicine.

In Xenopus laevis oocytes a mitochondrial cloud (MC) is found between the nucleus and the plasma membrane at stages I–II of oogenesis. The MC contains RNAs that are transported to the future vegetal pole at stage II of oogenesis. In particular, germinal plasm mRNAs are found in the Message Transport Organiser (METRO) region, the MC region opposite to the nucleus. At stages II–III, a second pathway transports Vg1 and VegT mRNAs to the area where the MC content merges with the vegetal cortex. Microtubules become polarized at the sites of migration of Vg1 and VegT mRNAs through an unknown signalling mechanism. In early meiotic stages, the centrioles are almost completely lost with their remnants being dispersed into the cytoplasm and the MC, which may contain a MTOC to be used in the later localization pathway of the mRNAs. In mammals, XNOA 36 encodes a member of a highly conserved protein family and localises to the nucleolus or in the centromeres. In the Xenopus late stage I oocyte, XNOA 36 mRNA is transiently segregated in one half of the oocyte, anchored by a cytoskeletal network that contains spectrin. Here we found that XNOA 36 transcript also localises to the nucleoli and in the METRO region. XNOA 36 protein immunolocalization, using an antibody employed for the library immunoscreening that depicted XNOA 36 expression colonies, labels the migrating MC, the cytoplasm of stage I oocytes and in particular the vegetal cortex facing the MC. The possible role of XNOA 36 in mRNA anchoring to the vegetal cortex or in participating in early microtubule reorganization is discussed.

Neospora caninum is a recently identified coccidian parasite which was, until 1988, misdiagnosed as Toxoplasma gondii. It causes paralysis and death in dogs and neonatal mortality and abortion in cattle, sheep, goats and horses. The life-cycle of Neospora has not yet been elucidated. The only two stages identified so far are tissue cysts and intracellularly dividing tachyzoites. Very little is known about the biology of this species. We have set up a fluorescence-based adhesion/invasion assay in order to investigate the interaction of N. caninum tachyzoites with bovine aorta endothelial (BAE) cells in vitro. Treatment of both host cells and parasites with metabolic inhibitors determined the metabolic requirements for adhesion and invasion. Chemical and enzymatic modifications of parasite and endothelial cell surfaces were used in order to obtain information on the nature of cell surface components responsible for the interaction between parasite and host. Electron microscopical investigations defined the ultrastructural characteristics of the adhesion and invasion process, and provided information on the intracellular development of the parasites.

A three-dimensional (3D) reconstruction of the cytoskeleton and a clathrin-coated pit in mammalian cells has been achieved from a focal-series of images recorded in an aberration-corrected scanning transmission electron microscope (STEM). The specimen was a metallic replica of the biological structure comprising Pt nanoparticles 2–3 nm in diameter, with a high stability under electron beam radiation. The 3D dataset was processed by an automated deconvolution procedure. The lateral resolution was 1.1 nm, set by pixel size. Particles differing by only 10 nm in vertical position were identified as separate objects with greater than 20% dip in contrast between them. We refer to this value as the axial resolution of the deconvolution or reconstruction, the ability to recognize two objects, which were unresolved in the original dataset. The resolution of the reconstruction is comparable to that achieved by tilt-series transmission electron microscopy. However, the focal-series method does not require mechanical tilting and is therefore much faster. 3D STEM images were also recorded of the Golgi ribbon in conventional thin sections containing 3T3 cells with a comparable axial resolution in the deconvolved dataset.

Cell motility is an integral part of a diverse set of biological processes. The quest formathematical models of cell motility has prompted the development of automated approachesfor gathering quantitative data on cell morphology, and the distribution of molecularplayers involved in cell motility. Here we review recent approaches for quantifying cellmotility, including automated cell segmentation and tracking. Secondly, we present our ownnovel method for tracking cell boundaries of moving cells, the Electrostatic ContourMigration Method (ECMM), as an alternative to the generally accepted level set method(LSM). ECMM smoothly tracks regions of the cell boundary over time to compute localmembrane displacements using the simple underlying concept of electrostatics. It offerssubstantial speed increases and reduced computational overheads in comparison to the LSM.We conclude with general considerations regarding boundary tracking in the context ofmathematical modelling.

We have used antibodies raised against a cytoskeletal protein, microtubule-associated protein 1A (MAP 1A), to stain adult rat retina. In cryostat and polyethylene glycol-embedded radial sections, the fiber layer, ganglion cell layer, and inner plexiform layer were highly immunoreactive, a finding that suggested that the ganglion cell somata, axons, and dendrites were recognized by these antibodies. Retrograde labeling of retinal cell somata from the superior colliculus and dorso-lateral geniculate nucleus to identify ganglion cells showed colocalization of the tracer and immunoreactive cells. Double labeling with nuclear stains revealed that many cells in the ganglion cell layer, which are likely displaced amacrine cells, were not recognized by these antibodies. Furthermore, transection of ganglion cell axons, a procedure that causes retrograde degeneration of many of the axotomized ganglion cells, led to a decrease in the number of anti-MAP 1A immunoreactive cells in retinal wholemounts. Thus, MAP 1A antibodies preferentially stain ganglion cell somata and dendrites but not amacrine cells. These antibodies should be useful ganglion cell markers.