The cortical cytoskeletal network and cell-wall dynamics in the unicellular charophycean green alga Penium margaritaceum

Background and Aims

Penium margaritaceum is a unicellular charophycean green alga with a unique bi-directional polar expansion mechanism that occurs at the central isthmus zone prior to cell division. This entails the focused deposition of cell-wall polymers coordinated by the activities of components of the endomembrane system and cytoskeletal networks. The goal of this study was to elucidate the structural organization of the cortical cytoskeletal network during the cell cycle and identify its specific functional roles during key cell-wall developmental events: pre-division expansion and cell division.

Methods

Microtubules and actin filaments were labelled during various cell cycle phases with an anti-tubulin antibody and rhodamine phalloidin, respectively. Chemically induced disruption of the cytoskeleton was used to elucidate specific functional roles of microtubules and actin during cell expansion and division. Correlation of cytoskeletal dynamics with cell-wall development included live cell labelling with wall polymer-specific antibodies and electron microscopy.

Key Results

The cortical cytoplasm of Penium is highlighted by a band of microtubules found at the cell isthmus, i.e. the site of pre-division wall expansion. This band, along with an associated, transient band of actin filaments, probably acts to direct the deposition of new wall material and to mark the plane of the future cell division. Two additional bands of microtubules, which we identify as satellite bands, arise from the isthmus microtubular band at the onset of expansion and displace toward the poles during expansion, ultimately marking the isthmus of future daughter cells. Treatment with microtubule and actin perturbation agents reversibly stops cell division.

Conclusions

The cortical cytoplasm of Penium contains distinct bands of microtubules and actin filaments that persist through the cell cycle. One of these bands, termed the isthmus microtubule band, or IMB, marks the site of both pre-division wall expansion and the zone where a cross wall will form during cytokinesis. This suggests that prior to the evolution of land plants, a dynamic, cortical cytoskeletal array similar to a pre-prophase band had evolved in the charophytes. However, an interesting variation on the cortical band theme is present in Penium, where two satellite microtubule bands are produced at the onset of cell expansion, each of which is destined to become an IMB in the two daughter cells after cytokinesis. These unique cytoskeletal components demonstrate the close temporal control and highly coordinated cytoskeletal dynamics of cellular development in Penium.     

GSK3 regulates mitotic chromosomal alignment through CRMP4

Background

Glycogen Synthase Kinase 3 (GSK3) has been implicated in regulating chromosomal alignment and mitotic progression but the physiological substrates mediating these GSK3-dependent effects have not been identified. Collapsin Response Mediator Protein 4 (CRMP4) is a cytosolic phosphoprotein known to regulate cytoskeletal dynamics and is a known physiological substrate of GSK3. In this study, we investigate the role of CRMP4 during mitosis.

Methodology and Principal Findings

Here we demonstrate that during mitosis CRMP4 phosphorylation is regulated in a GSK3-dependent manner. We show that CRMP4 localizes to spindle microtubules during mitosis and loss of CRMP4 disrupts chromosomal alignment and mitotic progression. The effect of CRMP4 on chromosomal alignment is dependent on phosphorylation by GSK3 identifying CRMP4 as a critical GSK3 substrate during mitotic progression. We also provide mechanistic data demonstrating that CRMP4 regulates spindle microtubules consistent with its known role in the regulation of the microtubule cytoskeleton.

Conclusion and Significance

Our findings identify CRMP4 as a key physiological substrate of GSK3 in regulating chromosomal alignment and mitotic progression through its effect on spindle microtubules.     

The actin-binding protein capulet genetically interacts with the microtubule motor kinesin to maintain neuronal dendrite homeostasis

Background

Neurons require precise cytoskeletal regulation within neurites, containing microtubule tracks for cargo transport in axons and dendrites or within synapses containing organized actin. Due to the unique architecture and specialized function of neurons, neurons are particularly susceptible to perturbation of the cytoskeleton. Numerous actin-binding proteins help maintain proper cytoskeletal regulation.

Methodology/Principal Findings

From a Drosophila forward genetic screen, we identified a mutation in capulet-encoding a conserved actin-binding protein-that causes abnormal aggregates of actin within dendrites. Through interaction studies, we demonstrate that simultaneous genetic inactivation of capulet and kinesin heavy chain, a microtubule motor protein, produces elongate cofilin-actin rods within dendrites but not axons. These rods resemble actin-rich structures induced in both mammalian neurodegenerative and Drosophila Alzheimer''s models, but have not previously been identified by loss of function mutations in vivo. We further demonstrate that mitochondria, which are transported by Kinesin, have impaired distribution along dendrites in a capulet mutant. While Capulet and Cofilin may biochemically cooperate in certain circumstances, in neuronal dendrites they genetically antagonize each other.

Conclusions/Significance

The present study is the first molecularly defined loss of function demonstration of actin-cofilin rods in vivo. This study suggests that simultaneous, seemingly minor perturbations in neuronal dendrites can synergize producing severe abnormalities affecting actin, microtubules and mitochondria/energy availability in dendrites. Additionally, as >90% of Alzheimer''s and Parkinson''s cases are sporadic this study suggests mechanisms by which multiple mutations together may contribute to neurodegeneration instead of reliance on single mutations to produce disease.     

An SK3 channel/nWASP/Abi-1 complex is involved in early neurogenesis

Background

The stabilization or regulated reorganization of the actin cytoskeleton is essential for cellular structure and function. Recently, we could show that the activation of the SK3-channel that represents the predominant SK-channel in neural stem cells, leads to a rapid local outgrowth of long filopodial processes. This observation indicates that the rearrangement of the actin based cytoskeleton via membrane bound SK3-channels might selectively be controlled in defined micro compartments of the cell.

Principal Findings

We found two important proteins for cytoskeletal rearrangement, the Abelson interacting protein 1, Abi-1 and the neural Wiskott Aldrich Syndrome Protein, nWASP, to be in complex with SK3- channels in neural stem cells (NSCs). Moreover, this interaction is also found in spines and postsynaptic compartments of developing primary hippocampal neurons and regulates neurite outgrowth during early phases of differentiation. Overexpression of the proteins or pharmacological activation of SK3 channels induces obvious structural changes in NSCs and hippocampal neurons. In both neuronal cell systems SK3 channels and nWASP act synergistic by strongly inducing filopodial outgrowth while Abi-1 behaves antagonistic to its interaction partners.

Conclusions

Our results give good evidence for a functional interplay of a trimeric complex that transforms incoming signals via SK3-channel activation into the local rearrangement of the cytoskeleton in early steps of neuronal differentiation involving nWASP and Abi-1 actin binding proteins.     

Modulation of phosducin-like protein 3 (PhLP3) levels promotes cytoskeletal remodelling in a MAPK and RhoA-dependent manner

Background

Phosducin-like protein 3 (PhLP3) forms a ternary complex with the ATP-dependent molecular chaperone CCT and its folding client tubulin. In vitro studies suggest PhLP3 plays an inhibitory role in β-tubulin folding while conversely in vivo genetic studies suggest PhLP3 is required for the correct folding of β-tubulin. We have a particular interest in the cytoskeleton, its chaperones and their role in determining cellular phenotypes associated with high level recombinant protein expression from mammalian cell expression systems.

Methodology/Principal Findings

As studies into PhLP3 function have been largely carried out in non mammalian systems, we examined the effect of human PhLP3 over-expression and siRNA silencing using a single murine siRNA on both tubulin and actin systems in mammalian Chinese hamster ovary (CHO) cell lines. We show that over-expression of PhLP3 promotes an imbalance of α and β tubulin subunits, microtubule disassembly and cell death. In contrast, β-actin levels are not obviously perturbed. On-the-other-hand, RNA silencing of PhLP3 increases RhoA-dependent actin filament formation and focal adhesion formation and promotes a dramatic elongated fibroblast-like change in morphology. This was accompanied by an increase in phosphorylated MAPK which has been associated with promoting focal adhesion assembly and maturation. Transient overexpression of PhLP3 in knockdown experiments rescues cells from the morphological change observed during PhLP3 silencing but mitosis is perturbed, probably reflecting a tipping back of the balance of PhLP3 levels towards the overexpression state.

Conclusions

Our results support the hypothesis that PhLP3 is important for the maintenance of β-tubulin levels in mammalian cells but also that its modulation can promote actin-based cytoskeletal remodelling by a mechanism linked with MAPK phosphorylation and RhoA-dependent changes. PhLP3 levels in mammalian cells are thus finely poised and represents a novel target for engineering industrially relevant cell lines to evolve lines more suited to suspension or adherent cell growth.     

LKB1 destabilizes microtubules in myoblasts and contributes to myoblast differentiation

Background

Skeletal muscle myoblast differentiation and fusion into multinucleate myotubes is associated with dramatic cytoskeletal changes. We find that microtubules in differentiated myotubes are highly stabilized, but premature microtubule stabilization blocks differentiation. Factors responsible for microtubule destabilization in myoblasts have not been identified.

Findings

We find that a transient decrease in microtubule stabilization early during myoblast differentiation precedes the ultimate microtubule stabilization seen in differentiated myotubes. We report a role for the serine-threonine kinase LKB1 in both microtubule destabilization and myoblast differentiation. LKB1 overexpression reduced microtubule elongation in a Nocodazole washout assay, and LKB1 RNAi increased it, showing LKB1 destabilizes microtubule assembly in myoblasts. LKB1 levels and activity increased during myoblast differentiation, along with activation of the known LKB1 substrates AMP-activated protein kinase (AMPK) and microtubule affinity regulating kinases (MARKs). LKB1 overexpression accelerated differentiation, whereas RNAi impaired it.

Conclusions

Reduced microtubule stability precedes myoblast differentiation and the associated ultimate microtubule stabilization seen in myotubes. LKB1 plays a positive role in microtubule destabilization in myoblasts and in myoblast differentiation. This work suggests a model by which LKB1-induced microtubule destabilization facilitates the cytoskeletal changes required for differentiation. Transient destabilization of microtubules might be a useful strategy for enhancing and/or synchronizing myoblast differentiation.     

Moderation of calpain activity promotes neovascular integration and lumen formation during VEGF-induced pathological angiogenesis

Background

Successful neovascularization requires that sprouting endothelial cells (ECs) integrate to form new vascular networks. However, architecturally defective, poorly integrated vessels with blind ends are typical of pathological angiogenesis induced by vascular endothelial growth factor-A (VEGF), thereby limiting the utility of VEGF for therapeutic angiogenesis and aggravating ischemia-related pathologies. Here we investigated the possibility that over-exuberant calpain activity is responsible for aberrant VEGF neovessel architecture and integration. Calpains are a family of intracellular calcium-dependent, non-lysosomal cysteine proteases that regulate cellular functions through proteolysis of numerous substrates.

Methodology/Principal Findings

In a mouse skin model of VEGF-driven angiogenesis, retroviral transduction with dominant-negative (DN) calpain-I promoted neovessel integration and lumen formation, reduced blind ends, and improved vascular perfusion. Moderate doses of calpain inhibitor-I improved VEGF-driven angiogenesis similarly to DN calpain-I. Conversely, retroviral transduction with wild-type (WT) calpain-I abolished neovessel integration and lumen formation. In vitro, moderate suppression of calpain activity with DN calpain-I or calpain inhibitor-I increased the microtubule-stabilizing protein tau in endothelial cells (ECs), increased the average length of microtubules, increased actin cable length, and increased the interconnectivity of vascular cords. Conversely, WT calpain-I diminished tau, collapsed microtubules, disrupted actin cables, and inhibited integration of cord networks. Consistent with the critical importance of microtubules for vascular network integration, the microtubule-stabilizing agent taxol supported vascular cord integration whereas microtubule dissolution with nocodazole collapsed cord networks.

Conclusions/Significance

These findings implicate VEGF-induction of calpain activity and impairment of cytoskeletal dynamics in the failure of VEGF-induced neovessels to form and integrate properly. Accordingly, calpain represents an important target for rectifying key vascular defects associated with pathological angiogenesis and for improving therapeutic angiogenesis with VEGF.     

Highly Dynamic Microtubules Improve the Effectiveness of Early Stages of Human Influenza A/NWS/33 Virus Infection in LLC-MK2 Cells

Background

This study aims to investigate the role of microtubule dynamics in the initiation of NWS/33 human influenza A (NWS) virus infection in MDCK and LLC-MK2 mammalian kidney cells. We previously demonstrated a host-dependent role of the actin cytoskeleton in inducing restriction during the early phases of NWS infection. Furthermore, we showed the differential infectious entry of NWS virus in the above mentioned cell models.

Methodology/Principal Findings

By first employing a panel of microtubule-modulators, we evidenced that microtubule-stabilization negatively interferes with NWS replication in LLC-MK2 but not in MDCK cells. Conversely, microtubule-depolymerization improves NWS growth in LLC-MK2 but not in the MDCK model. By using immunofluorescence labelling and Western blotting analyses upon NWS infection in mammalian kidney cells, it was observed that the occurrence of alpha-tubulin hyperacetylation - a post-translational modified form suggestive of stable microtubules - was significantly delayed in LLC-MK2 when compared to MDCK cells. Furthermore, mock-infected LLC-MK2 cells were shown to have higher levels of both acetylated alpha-tubulin and microtubule-associated protein 4 (MAP4), the latter being essential for the maintenance of normal microtubule polymer levels in interphase epithelial cells. Finally, to obtain highly dynamic microtubules in LLC-MK2 cells, we knocked down the expression of MAP4 by using a RNA-mediated RNA interference approach. The results evidenced that MAP4 silencing improves NWS growth in LLC-MK2 cells.

Conclusion

By evidencing the cell type-dependent regulatory role of microtubule dynamics on NWS replication in mammalian kidney cells, we demonstrated that microtubule-stabilization represents a restriction factor for the initiation of NWS infection in LLC-MK2 but not in MDCK cells.     

Proteins implicated in mediating self-incompatibility-induced alterations to the actin cytoskeleton of Papaver pollen

Background and Aims

Sexual reproduction in angiosperms involves a network of signalling and interactions between pollen and pistil. To promote out-breeding, an additional layer of interactions, involving self-incompatibility (SI), is used to prevent self-fertilization. SI is generally controlled by the S-locus, and comprises allelic pollen and pistil S-determinants. This provides the basis of recognition, and consequent rejection, of incompatible pollen. In Papaver rhoeas, SI involves interaction of pistil PrsS and pollen PrpS, triggering a Ca²⁺-dependent signalling network. This results in rapid and distinctive alterations to both the actin and microtubule cytoskeleton being triggered in ‘self’ pollen. Some of these alterations are implicated in mediating programmed cell death, involving activation of several caspase-like proteases.

Scope

Here we review and discuss our current understanding of the cytoskeletal alterations induced in incompatible pollen during SI and their relationship with programmed cell death. We focus on data relating to the formation of F-actin punctate foci, which have, to date, not been well characterized. The identification of two actin-binding proteins that interact with these structures are reviewed. Using an approach that enriched for F-actin from SI-induced pollen tubes using affinity purification followed by mass spectrometry, further proteins were identified as putative interactors with the F-actin foci in an SI situation.

Key Results

Previously two important actin-binding proteins, CAP and ADF, had been identified whose localization altered with SI, both showing co-localization with the F-actin punctate foci based on immunolocalization studies. Further analysis has identified differences between proteins associated with F-actin from SI-induced pollen samples and those associated with F-actin in untreated pollen. This provides candidate proteins implicated in either the formation or stabilization of the punctate actin structures formed during SI.

Conclusions

This review brings together for the first time, our current understanding of proteins and events involved in SI-induced signalling to the actin cytoskeleton in incompatible Papaver pollen.     

Conservation of tubulin-binding sequences in TRPV1 throughout evolution

Background

Transient Receptor Potential Vanilloid sub type 1 (TRPV1), commonly known as capsaicin receptor can detect multiple stimuli ranging from noxious compounds, low pH, temperature as well as electromagnetic wave at different ranges. In addition, this receptor is involved in multiple physiological and sensory processes. Therefore, functions of TRPV1 have direct influences on adaptation and further evolution also. Availability of various eukaryotic genomic sequences in public domain facilitates us in studying the molecular evolution of TRPV1 protein and the respective conservation of certain domains, motifs and interacting regions that are functionally important.

Methodology and Principal Findings

Using statistical and bioinformatics tools, our analysis reveals that TRPV1 has evolved about ∼420 million years ago (MYA). Our analysis reveals that specific regions, domains and motifs of TRPV1 has gone through different selection pressure and thus have different levels of conservation. We found that among all, TRP box is the most conserved and thus have functional significance. Our results also indicate that the tubulin binding sequences (TBS) have evolutionary significance as these stretch sequences are more conserved than many other essential regions of TRPV1. The overall distribution of positively charged residues within the TBS motifs is conserved throughout evolution. In silico analysis reveals that the TBS-1 and TBS-2 of TRPV1 can form helical structures and may play important role in TRPV1 function.

Conclusions and Significance

Our analysis identifies the regions of TRPV1, which are important for structure – function relationship. This analysis indicates that tubulin binding sequence-1 (TBS-1) near the TRP-box forms a potential helix and the tubulin interactions with TRPV1 via TBS-1 have evolutionary significance. This interaction may be required for the proper channel function and regulation and may also have significance in the context of Taxol®-induced neuropathy.     

Actin Turnover Is Required for Myosin-Dependent Mitochondrial Movements in Arabidopsis Root Hairs

Background

Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements.

Methodology/Principal Findings

We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP-FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria.

Conclusions/Significance

Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events.     

Dynamic Subunit Exchange and the Regulation of Microtubule Assembly by the Stress Response Protein Human αB Crystallin

Background

The small heat shock protein (sHSP), human αB crystallin, forms large, polydisperse complexes that modulate the tubulin-microtubule equilibrium using a dynamic mechanism that is poorly understood. The interactive sequences in αB crystallin for tubulin are surface exposed, and correspond to interactive sites for the formation of αB crystallin complexes.

Methodology/Principal Findings

There is sequence homology between tubulin and the interactive domains in the β8-strand of the core domain and the C-terminal extension of αB crystallin. This study investigated the hypothesis that the formation of tubulin and αB crystallin quaternary structures was regulated through common interactive domains that alter the dynamics of their assembly. Size exclusion chromatography (SEC), SDS-PAGE, microtubule assembly assays, aggregation assays, multiple sequence alignment, and molecular modeling characterized the dynamic response of tubulin assembly to increasing concentrations of αB crystallin. Low molar ratios of αB crystallin∶tubulin were favorable for microtubule assembly and high molar ratios of αB crystallin∶tubulin were unfavorable for microtubule assembly. Interactions between αB crystallin and unassembled tubulin were observed using SEC and SDS-PAGE.

Conclusions/Significance

Subunits of αB crystallin that exchange dynamically with the αB crystallin complex can interact with tubulin subunits to regulate the equilibrium between tubulin and microtubules.     

Role of spectraplakin in Drosophila photoreceptor morphogenesis

Background

Crumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the apical membrane domain and adherens junction during Drosophila photoreceptor morphogenesis. It has recently been found that stable microtubules in developing Drosophila photoreceptors were linked to Crb localization. Coordinated interactions between microtubule and actin cytoskeletons are involved in many polarized cellular processes. Since Spectraplakin is able to bind both microtubule and actin cytoskeletons, the role of Spectraplakin was analyzed in the regulations of apical Crb domain in developing Drosophila photoreceptors.

Methodology/Principal Findings

The localization pattern of Spectraplakin in developing pupal photoreceptors showed a unique intracellular distribution. Spectraplakin localized at rhabdomere terminal web which is at the basal side of the apical Crb or rhabdomere, and in between the adherens junctions. The spectraplakin mutant photoreceptors showed dramatic mislocalizations of Crb, adherens junctions, and the stable microtubules. This role of Spectraplakin in Crb and adherens junction regulation was further supported by spectraplakin''s gain-of-function phenotype. Spectraplakin overexpression in photoreceptors caused a cell polarity defect including dramatic mislocalization of Crb, adherens junctions and the stable microtubules in the developing photoreceptors. Furthermore, a strong genetic interaction between spectraplakin and crb was found using a genetic modifier test.

Conclusions/Significance

In summary, we found a unique localization of Spectraplakin in photoreceptors, and identified the role of spectraplakin in the regulation of the apical Crb domain and adherens junctions through genetic mutational analysis. Our data suggest that Spectraplakin, an actin-microtubule cross-linker, is essential in the apical and adherens junction controls during the photoreceptors morphogenesis.     

Potent Antiproliferative Cembrenoids Accumulate in Tobacco upon Infection with Rhodococcus fascians and Trigger Unusual Microtubule Dynamics in Human Glioblastoma Cells

Aims

Though plant metabolic changes are known to occur during interactions with bacteria, these were rarely challenged for pharmacologically active compounds suitable for further drug development. Here, the occurrence of specific chemicals with antiproliferative activity against human cancer cell lines was evidenced in hyperplasia (leafy galls) induced when plants interact with particular phytopathogens, such as the Actinomycete Rhodococcus fascians.

Methods

We examined leafy galls fraction F3.1.1 on cell proliferation, cell division and cytoskeletal disorganization of human cancer cell lines using time-lapse videomicroscopy imaging, combined with flow cytometry and immunofluorescence analysis. We determined the F3.1.1-fraction composition by gas chromatography coupled to mass spectrometry.

Results

The leafy galls induced on tobacco by R. fascians yielded fraction F3.1.1 which inhibited proliferation of glioblastoma U373 cells with an IC₅₀ of 4.5 µg/mL, F.3.1.1 was shown to increase cell division duration, cause nuclear morphological deformations and cell enlargement, and, at higher concentrations, karyokinesis defects leading to polyploidization and apoptosis. F3.1.1 consisted of a mixture of isomers belonging to the cembrenoids. The cellular defects induced by F3.1.1 were caused by a peculiar cytoskeletal disorganization, with the occurrence of fragmented tubulin and strongly organized microtubule aggregates within the same cell. Colchicine, paclitaxel, and cembrene also affected U373 cell proliferation and karyokinesis, but the induced microtubule rearrangement was very different from that provoked by F3.1.1. Altogether our data indicate that the cembrenoid isomers in F3.1.1 have a unique mode of action and are able to simultaneously modulate microtubule polymerization and stability.     

Actomyosin-dependent cortical dynamics contributes to the prophase force-balance in the early Drosophila embryo

Background

The assembly of the Drosophila embryo mitotic spindle during prophase depends upon a balance of outward forces generated by cortical dynein and inward forces generated by kinesin-14 and nuclear elasticity. Myosin II is known to contribute to the dynamics of the cell cortex but how this influences the prophase force-balance is unclear.

Principal Findings

Here we investigated this question by injecting the myosin II inhibitor, Y27632, into early Drosophila embryos. We observed a significant increase in both the area of the dense cortical actin caps and in the spacing of the spindle poles. Tracking of microtubule plus ends marked by EB1-GFP and of actin at the cortex revealed that astral microtubules can interact with all regions of these expanded caps, presumably via their interaction with cortical dynein. In Scrambled mutants displaying abnormally small actin caps but normal prophase spindle length in late prophase, myosin II inhibition produced very short spindles.

Conclusions

These results suggest that two complementary outward forces are exerted on the prophase spindle by the overlying cortex. Specifically, dynein localized on the mechanically firm actin caps and the actomyosin-driven contraction of the deformable soft patches of the actin cortex, cooperate to pull astral microtubules outward. Thus, myosin II controls the size and dynamic properties of the actin-based cortex to influence the spacing of the poles of the underlying spindle during prophase.     

The Actin Targeting Compound Chondramide Inhibits Breast Cancer Metastasis via Reduction of Cellular Contractility

Background

A major player in the process of metastasis is the actin cytoskeleton as it forms key structures in both invasion mechanisms, mesenchymal and amoeboid migration. We tested the actin binding compound Chondramide as potential anti-metastatic agent.

Methods

In vivo, the effect of Chondramide on metastasis was tested employing a 4T1-Luc BALB/c mouse model. In vitro, Chondramide was tested using the highly invasive cancer cell line MDA-MB-231 in Boyden-chamber assays, fluorescent stainings, Western blot and Pull down assays. Finally, the contractility of MDA-MB-231 cells was monitored in 3D environment and analyzed via PIV analysis.

Results

In vivo, Chondramide treatment inhibits metastasis to the lung and the migration and invasion of MDA-MB-231 cells is reduced by Chondramide in vitro. On the signaling level, RhoA activity is decreased by Chondramide accompanied by reduced MLC-2 and the stretch induced guanine nucleotide exchange factor Vav2 activation. At same conditions, EGF-receptor autophosphorylation, Akt and Erk as well as Rac1 are not affected. Finally, Chondramide treatment disrupted the actin cytoskeleton and decreased the ability of cells for contraction.

Conclusions

Chondramide inhibits cellular contractility and thus represents a potential inhibitor of tumor cell invasion.     

Intact microtubules preserve transient receptor potential vanilloid 1 (TRPV1) functionality through receptor binding

The transient receptor potential cation channel subfamily V member 1 (TRPV1) is a protein currently under scrutiny as a pharmacological target for pain management therapies. Recently, the role of TRPV1-microtubule interaction in transducing nociception stimuli to cells by cytoskeletal rearrangement was proposed. In this work, we investigate TRPV1-microtubule interaction in living cells under the resting or activated state of TRPV1, as well as in presence of structurally intact or depolymerized cytoskeletal microtubules. We combined a toolbox of high resolution/high sensitivity fluorescence imaging techniques (such as FRET, correlation spectroscopy, and fluorescence anisotropy) to monitor TRPV1 aggregation status, membrane mobility, and interaction with microtubules. We found that TRPV1 is a dimeric membrane protein characterized by two populations with different diffusion properties in basal condition. After stimulation with resiniferatoxin, TRPV1 dimers tetramerize. The tetramers and the slower population of TRPV1 dimers bind dynamically to intact microtubules but not to tubulin dimers. Upon microtubule disassembly, the interaction with TRPV1 is lost thereby inducing receptor self-aggregation with partial loss of functionality. Intact microtubules play an essential role in maintaining TRPV1 functionality toward activation stimuli. This previously undisclosed property mirrors the recently reported role of TRPV1 in modulating microtubule assembly/disassembly and suggests the participation of these two players in a feedback cycle linking nociception and cytoskeletal remodeling.     

Down Regulation of Genes Involved in T Cell Polarity and Motility during the Induction of Heart Allograft Tolerance by Allochimeric MHC I

Background

The allochimeric MHC class I molecule [α1h1/u]-RT1.Aa that contains donor-type (Wistar Furth, WF; RT1u) epitopes displayed on recipient-type (ACI, RT1a) administered in conjunction with sub-therapeutic dose of cyclosporine (CsA) induces indefinite survival of heterotopic cardiac allografts in rat model. In vascularized transplantation models, the spleen contributes to graft rejection by generating alloantigen reactive T cells. The immune response in allograft rejection involves a cascade of molecular events leading to the formation of immunological synapses between T cells and the antigen-presenting cells.

Methodology/Principal Findings

To elucidate the molecular pathways involved in the immunosuppressive function of allochimeric molecule we performed microarray and quantitative RTPCR analyses of gene expression profile of splenic T cells from untreated, CsA treated, and allochimeric molecule + subtherapeutic dose of CsA treated animals at day 1, 3 and 7 of post transplantation. Allochimeric molecule treatment caused down regulation of genes involved in actin filament polymerization (RhoA and Rac1), cell adhesion (Catna1, Vcam and CD9), vacuolar transport (RhoB, Cln8 and ATP6v1b2), and MAPK pathway (Spred1 and Dusp6) involved in tubulin cytoskeleton reorganization and interaction between actin and microtubule cytoskeleton. All these genes are involved in T cell polarity and motility, i.e., their ability to move, scan and to form functional immunological synapse with antigen presenting cells (APCs).

Conclusions

These results indicate that the immunosuppressive function of allochimeric molecule may depend on the impairment of T cells'' movement and scanning ability, and possibly also the formation of immunological synapse. We believe that these novel findings may have important clinical implications for organ transplantation.