IQGAP1 regulation and roles in cancer

IQGAP1 is a key mediator of several distinct cellular processes, in particular cytoskeletal rearrangements. Recent studies have implicated a potential role for IQGAP1 in cancer, supported by the over-expression and distinct membrane localisation of IQGAP1 observed in a range of tumours. IQGAP1 is thought to contribute to the transformed cancer cell phenotype by regulating signalling pathways involved in cell proliferation and transformation, weakening of cell:cell adhesion contacts and stimulation of cell motility and invasion. In this review we discuss these different functional and regulatory roles of IQGAP1 and its homologues in relation to their potential impact on tumourigenesis.     

Regulation of β-catenin trafficking to the membrane in living cells

β-catenin is a key mediator of the Wnt signaling process and accumulates in the nucleus and at the membrane in response to Wnt-mediated inhibition of GSK-3β. In this study we used live cell photobleaching experiments to determine the dynamics and rate of recruitment of β-catenin at membrane adherens junctions (cell adhesion) and membrane ruffles (cell migration). First, we confirmed the nuclear-cytoplasmic shuttling of GFP-tagged β-catenin, and found that a small mobile pool of β-catenin can move from the nucleus to membrane ruffles in NIH 3T3 fibroblasts with a t_0.5 of ~ 30 s. Thus, β-catenin can shuttle between the nucleus and plasma membrane. The localized recruitment of β-catenin-GFP to membrane ruffles was more rapid, and the strong recovery observed after bleaching (mobile fraction 53%, t_0.5 ~5 s) is indicative of high turnover and transient association. In contrast, β-catenin-GFP displayed poor recovery at adherens junctions in MDCK epithelial cells (mobile fraction 10%, t_0.5 ~8 s), indicating stable retention at these membrane structures. We previously identified IQGAP1 as an upstream regulator of β-catenin at the membrane, and this is supported by photobleaching assays which now reveal IQGAP1 to be more stably anchored at membrane ruffles than β-catenin. Further analysis showed that LiCl-mediated inactivation of the kinase GSK-3β increased β-catenin membrane ruffle staining; this correlated with a faster rate of recruitment and not increased membrane retention of β-catenin. In summary, β-catenin displays a high turnover rate at membrane ruffles consistent with its dynamic internalization and recycling at these sites by macropinocytosis.     

The Respiratory Syncytial Virus Matrix Protein Possesses a Crm1-Mediated Nuclear Export Mechanism

The respiratory syncytial virus (RSV) matrix (M) protein is localized in the nucleus of infected cells early in infection but is mostly cytoplasmic late in infection. We have previously shown that M localizes in the nucleus through the action of the importin β1 nuclear import receptor. Here, we establish for the first time that M''s ability to shuttle to the cytoplasm is due to the action of the nuclear export receptor Crm1, as shown in infected cells, and in cells transfected to express green fluorescent protein (GFP)-M fusion proteins. Specific inhibition of Crm1-mediated nuclear export by leptomycin B increased M nuclear accumulation. Analysis of truncated and point-mutated M derivatives indicated that Crm1-dependent nuclear export of M is attributable to a nuclear export signal (NES) within residues 194 to 206. Importantly, inhibition of M nuclear export resulted in reduced virus production, and a recombinant RSV carrying a mutated NES could not be rescued by reverse genetics. That this is likely to be due to the inability of a nuclear export deficient M to localize to regions of virus assembly is indicated by the fact that a nuclear-export-deficient GFP-M fails to localize to regions of virus assembly when expressed in cells infected with wild-type RSV. Together, our data suggest that Crm1-dependent nuclear export of M is central to RSV infection, representing the first report of such a mechanism for a paramyxovirus M protein and with important implications for related paramyxoviruses.The Pneumovirus respiratory syncytial virus (RSV) within the Paramyxoviridae family is the most common cause of lower-respiratory-tract disease in infants (7). The negative-sense single-strand RNA genome of RSV encodes two nonstructural and nine structural proteins, comprising the envelope glycoproteins (F, G, and SH), the nucleocapsid proteins (N, P, and L), the nucleocapsid-associated proteins (M2-1 and M2-2), and the matrix (M) protein (1, 7, 11). Previously, we have shown that M protein localizes in the nucleus at early stages of infection, but later in infection it is localized mainly in the cytoplasm, in association with nucleocapsid-containing cytoplasmic inclusions (13, 16). The M proteins of other negative-strand viruses, such as Sendai virus, Newcastle disease virus, and vesicular stomatitis virus (VSV), have also been observed in the nucleus at early stages of infection (32, 40, 48). Interestingly, the M proteins of all of these viruses, including RSV, play major roles in virus assembly, which take place in the cytoplasm and at the cell membrane (11, 12, 24, 34, 36, 39), but the mechanisms by which trafficking between the nucleus and cytoplasm occurs are unknown.The importin β family member Crm1 (exportin 1) is known to mediate nuclear export of proteins bearing leucine-rich nuclear export signals (NES) (8, 9, 18, 19, 37, 42, 43), such as the human immunodeficiency virus type 1 Rev protein (4). In the case of the influenza virus matrix (M1) protein, binding to the influenza virus nuclear export protein, which possesses a Crm1-recognized NES, appears to be responsible for its export from the nucleus, bound to the influenza virus RNA (3).We have recently shown that RSV M localizes in the nucleus through a conventional nuclear import pathway dependent on the nuclear import receptor importin β1 (IMPβ1) and the guanine nucleotide-binding protein Ran (14). In the present study, we show for the first time that RSV M possesses a Crm1-dependent nuclear export pathway, based on experiments using the specific inhibitor leptomycin B (LMB) (25), both in RSV-infected cells and in green fluorescent protein (GFP)-M fusion protein-expressing transfected cells. We use truncated and point-mutated M derivatives to map the Crm1-recognized NES within the M sequence and show that Crm1-dependent nuclear export is critical to the RSV infectious cycle, since LMB treatment early in infection, inhibiting M export from the nucleus, reduces RSV virion production and a recombinant RSV carrying a NES mutation in M was unable to replicate, probably because M deficient in nuclear export could not localize to areas of virus assembly, as shown in RSV-infected cells transfected to express GFP-M. This is the first report of a Crm1-mediated nuclear export pathway for a paramyxovirus M protein, with implications for the trafficking and function of other paramyxovirus M proteins.     

The history and composition of the Raymond A. Dart Collection of Human Skeletons at the University of the Witwatersrand,Johannesburg, South Africa

The Raymond A. Dart Collection of Human Skeletons (Dart Collection) is housed in the School of Anatomical Sciences at the University of the Witwatersrand, Johannesburg, South Africa, and comprises one of the largest documented cadaver‐derived human skeletal assemblages in the world. This collection originated in the early 1920s as a result of the efforts of Raymond Dart and continues to grow. The skeletons included represent varied indigenous and immigrant populations from southern Africa, Europe and Asia. This contribution documents the history of the collection and provides an updated inventory and demographic assessment of this valuable research collection. According to a recent inventory the Dart Collection currently comprises 2,605 skeletons representing individuals from regional SA African (76%), White (15%), Coloured (4%) and Indian (0.3%) populations. A large proportion of the skeletons (71%) represent males. The recorded ages at death range from the first year to over 100 years of age, but the majority of individuals died between the ages of 20 and 70. The Dart Collection has been affected by collection procedures based on availability. All of the cadavers collected before 1958, and large proportions subsequently, were derived from unclaimed bodies in regional South African hospitals. Some details of documentation (age at death, population group) are estimates and some aspects of the collection demographics (sex ratios) do not closely reflect any living South African population. Our inventory and analysis of the Dart Collection is aimed to assist researchers planning research on the materials from this collection. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

Light-dependent hypersensitive response and resistance signaling against Turnip Crinkle Virus in Arabidopsis

Resistance to Turnip Crinkle Virus (TCV) in Arabidopsis ecotype Dijon (Di)-17 is conferred by the resistance gene HRT and a recessive locus rrt. In Di-17, TCV elicits a hypersensitive response (HR), which is accompanied by increased expression of pathogenesis-related (PR) genes and high levels of salicylic acid (SA). We have previously shown that HRT-mediated resistance to TCV is dependent on SA-mediated signal transduction and that increased levels of SA confer enhanced resistance to TCV via upregulation of the HRT gene. Here we show that HRT-mediated HR and resistance are dependent on light. A dark treatment immediately following TCV inoculation suppressed HR, resistance and activation of the majority of the TCV-induced genes. However, the absence of light did not affect either TCV-induced elevated levels of free SA or the expression of HRT. Interestingly, in the dark, transgenic plants overexpressing HRT showed susceptibility, but overexpression of HRT coupled with high levels of endogenous SA resulted in pronounced resistance. Consistent with these results is the finding that exogenous application of SA prior to TCV inoculation partially overcame the requirement for light. Light was also required for N gene-mediated HR and resistance to Tobacco Mosaic Virus, suggesting that it is an important factor which may be generally required during defense signaling.     

A novel dermal tissue construct: Development and in vitro characterization

A dermal tissue construct composed of human dermal fibroblasts and a chitosan sponge has been developed, targeted towards the treatment of diabetic nonhealing ulcers. The construct has been designed in a way that the dermal fibroblasts are arranged as a three‐dimensional sheet adhered entirely on one side of the chitosan sponge. This design would allow maximal diffusion of growth factors from the cells to the wound bed when the construct is applied on the wound with the cellular sheet side making contact with the wound bed. The diffusion of secreted growth factors would take place directly from cells to the wound bed without being impeded by a matrix. The cells are present at a high density in the dermal construct, which would aid in accelerated wound healing. The construct has a porous chitosan sponge base, which would allow gas exchange, and renders the dermal construct very flexible so that it would take the shape of the wound contours well, while having mechanical integrity. The viability of cells in the construct is greater than 90%. The dermal construct produces a high amount of vascular endothelial growth factor, from 42 ng to 31 ng in 24 h. The construct also produces high amounts of Interleukin‐8 (IL‐8), from 375 ng to 1065 ng in the first 24 h. Both VEGF and IL‐8 have important roles in the healing of chronic diabetic ulcers. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Urban riparian systems function as corridors for both native and invasive plant species

Riparian areas are often the only green areas left in urban and suburban landscapes, providing opportunities for conservation and connectivity of both aquatic and terrestrial organisms. While city planners and land managers often tout the importance of riparian networks for these uses, it is not well established if urban riparian plant communities are actually functioning as connected assemblages. Furthermore, urban riparian zones are well known to be highly invaded by non-native plant species and may be functioning to increase the spread of non-native species across the landscape. Here we examine connectivity of plant assemblages in riparian networks within an extensively urbanized landscape. We sampled riparian plant communities at 13 sites along three second-order streams of the Rahway River watershed, New Jersey. We also characterized propagule dispersal at each site by sampling litter packs on the river banks five times between March–October 2011 and identifying germinants from litter packs after cold stratification. Species turnover of both riparian and litter vegetation was more strongly associated with flow distance, particularly for native species, indicating that riverine systems are important for promoting connectivity of native plant assemblages in urban landscapes. However, non-native germinants significantly dominated propagule dispersal along the stream reaches, particularly early in the growing season, suggesting spread utilizing the river system and preemption may be an important mechanism for invasion success in this system. Our data show that management of invasive species should be planned and implemented at the watershed scale to reduce spread via the river system.     

Rhizosphere competent <Emphasis Type="Italic">Pantoea agglomerans</Emphasis> enhances maize (<Emphasis Type="Italic">Zea mays</Emphasis>) and chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) growth,without altering the rhizosphere functional diversity

Plant growth promoting Pantoea agglomerans NBRISRM (NBRISRM) was able to produce 60.4 μg/ml indole acetic acid and solubilize 77.5 μg/ml tri-calcium phosphate under in vitro conditions. Addition of 2% NaCl (w/v) in the media induced the IAA production and phosphate solubilization by 11% and 7%, respectively. For evaluating the plant growth promotory effect of NBRISRM inoculation a micro plot trial was conducted using maize and chickpea as host plants. The results revealed significant increase in all growth parameters tested in NBRISRM inoculated maize and chickpea plants, which were further confirmed by higher macronutrients (N, P and K) accumulation as compared to un-inoculated controls. Throughout the growing season of maize and chickpea, rhizosphere population of NBRISRM were in the range 10⁷–10⁸ CFU/g soil and competing with 10⁷–10⁹ CFU/g soil with heterogeneous bacterial population. Functional richness, diversity, and evenness were found significantly higher in maize rhizosphere as compared to chickpea, whereas NBRISRM inoculation were not able to change it, in both crops as compared to their un-inoculated control. To the best of our knowledge this is first report where we demonstrated the effect of P. agglomerans strain for improving maize and chickpea growth without altering the functional diversity.     

Schlafen-3 decreases cancer stem cell marker expression and autocrine/juxtacrine signaling in FOLFOX-resistant colon cancer cells

We have previously demonstrated that expression of the novel gene schlafen-3 (Slfn-3) correlates with intestinal epithelial cell differentiation (Patel VB, Yu Y, Das JK, Patel BB, Majumdar AP. Biochem Biophys Res Commun 388: 752-756, 2009). The present investigation was undertaken to examine whether Slfn-3 plays a role in regulating differentiation of FOLFOX-resistant (5-fluorouracil + oxaliplatin) colon cancer cells that are highly enriched in cancer stem cells (CSCs). Transfection of Slfn-3 in FOLFOX-resistant colon cancer HCT-116 cells resulted in increase of alkaline phosphatase activity, a marker of intestinal differentiation. Additionally, Slfn-3 transfection resulted in reduction of mRNA and protein levels of the CSC markers CD44, CD133, CD166, and aldehyde dehydrogenase 1 in both FOLFOX-resistant HCT-116 and HT-29 cells. This was accompanied by decreased formation of tumorosphere/colonosphere (an in vitro model of tumor growth) in stem cell medium and inhibition of expression of the chemotherapeutic drug transporter protein ABCG2. Additionally, Slfn-3 transfection of FOLFOX-resistant HCT-116 and HT-29 cells reduced Hoechst 33342 dye exclusion. Finally, Slfn-3 transfection inhibited the expression of transforming growth factor-α in both FOLFOX-resistant colon cancer cells, but stimulated apoptosis in response to additional FOLFOX treatment. In summary, our data demonstrate that Slfn-3 expression inhibits multiple characteristics of CSC-enriched, FOLFOX-resistant colon cancer cells, including induction of differentiation and reduction in tumorosphere/colonosphere formation, drug transporter activity, and autocrine stimulation of proliferation. Thus Slfn-3 expression may render colon CSCs more susceptible to cancer chemotherapeutics.