Lifei Fan

The Identification of Lymphocyte-Like Cells and Lymphoid-Related Genes in Amphioxus Indicates the Twilight for the Emergency of Adaptive Immune System

To seek evidence of a primitive adaptive immune system (AIS) before vertebrate, we examined whether lymphocytes or lymphocyte-like cells and the related molecules participating in the lymphocyte function existed in amphioxus. Anatomical analysis by electron microscopy revealed the presence of lymphocyte-like cells in gills, and these cells underwent morphological changes in response to microbial pathogens that are reminiscent of those of mammalian lymphocytes executing immune response to microbial challenge. In addition, a systematic comparative analysis of our cDNA database of amphioxus identified a large number of genes whose vertebrate counterparts are involved in lymphocyte function. Among these genes, several genes were found to be expressed in the vicinity of the lymphocyte-like cells by in situ hybridization and up-regulated after exposure to microbial pathogens. Our findings in the amphioxus indicate the twilight for the emergency of AIS before the invertebrate-vertebrate transition during evolution.

The small GTPase Rif is an alternative trigger for the formation of actin stress fibers in epithelial cells

Actin stress fibers are fundamental components of the actin cytoskeleton that produce contractile force in non-muscle cells. The formation of stress fibers is controlled by the small GTPase RhoA and two highly related proteins, RhoB and RhoC. Together, this subgroup of actin-regulatory proteins represents the canonical pathway of stress-fiber formation. Here, we show that the Rif GTPase is an alternative trigger of stress-fiber formation in epithelial cells. Rif is distantly related to RhoA; however, we show that the two proteins share a common downstream partner in stress-fiber formation – the Diaphanous-related formin mDia1. Rif-induced stress fibers also depend on the activity of the ROCK protein kinase. Unlike RhoA, Rif does not raise ROCK activity in cells, instead Rif appears to regulate the localization of myosin light chain phosphorylation. This study establishes Rif as a general regulator of Diaphanous-related formins and shows how non-classical Rho family members can access classical Rho pathways to create new signaling interfaces in cytoskeletal regulation.

The primitive immune system of amphioxus provides insights into the ancestral structure of the vertebrate immune system.

Amphioxus is considered to be the basal chordate. However, the structural and anatomical features of the amphioxus immune system are still elusive. Here we report a profile of structural studies of the amphioxus gill and gut, the first line of defending against microbes, through optical and electron microscopy. The amphioxus gut and gill are characterized by the following morphological criteria compared with vertebrates: primary and secondary lymphoid-like tissue clustered in the gill, a thicker basement membrane with a large villus channel and lack of muscular layer in the gut, along with blood vessels that fill with phagocytes following microbial challenge. The phenomena of tissue repair after microbial invasion was observed, though no phagocytes were observed in the region of tissue necrosis. The epithelium cells of amphioxus gut showed active phagocytosis after the microbial challenge. A small number of free and fixed macrophage-like cells were also found in the amphioxus gut. The current results described the structure of the immune system and cellular defense against infection in a protochordate, which may help us in understanding the structural origin of the vertebrate immune system.

AspC-Mediated Aspartate Metabolism Coordinates the Escherichia coli Cell Cycle

Background The fast-growing bacterial cell cycle consists of at least two independent cycles of chromosome replication and cell division. To ensure proper cell cycles and viability, chromosome replication and cell division must be coordinated. It has been suggested that metabolism could affect the Escherichia coli cell cycle, but the idea is still lacking solid evidences. Methodology/Principle Findings We found that absence of AspC, an aminotransferase that catalyzes synthesis of aspartate, led to generation of small cells with less origins and slow growth. In contrast, excess AspC was found to exert the opposite effect. Further analysis showed that AspC-mediated aspartate metabolism had a specific effect in the cell cycle, as only extra aspartate of the 20 amino acids triggered production of bigger cells with more origins per cell and faster growth. The amount of DnaA protein per cell was found to be changed in response to the availability of AspC. Depletion of (p)ppGpp by ΔrelAΔspoT led to a slight delay in initiation of replication, but did not change the replication pattern found in the ΔaspC mutant. Conclusion/Significances The results suggest that AspC-mediated metabolism of aspartate coordinates the E. coli cell cycle through altering the amount of the initiator protein DnaA per cell and the division signal UDP-glucose. Furthermore, AspC sequence conservation suggests similar functions in other organisms.

The small Rho GTPase Rif and actin cytoskeletal remodelling

The Rif GTPase is a recent addition to small Rho GTPase family; it shares low homology with other members in the family and evolutionarily parallels with the development of vertebrates. Rif has the conserved Rho GTPase domain structures and cycles between a GDP-bound inactive form and a GTP-bound active form. In its active form, Rif signals through multiple downstream effectors. In the present review, our aim is to summarize the current information about the Rif effectors and how Rif remodels actin cytoskeleton in many aspects.

Neurotrophin Promotes Neurite Outgrowth by Inhibiting Rif GTPase Activation Downstream of MAPKs and PI3K Signaling

Members of the well-known semaphorin family of proteins can induce both repulsive and attractive signaling in neural network formation and their cytoskeletal effects are mediated in part by small guanosine 5’-triphosphatase (GTPases). The aim of this study was to investigate the cellular role of Rif GTPase in the neurotrophin-induced neurite outgrowth. By using PC12 cells which are known to cease dividing and begin to show neurite outgrowth responding to nerve growth factor (NGF), we found that semaphorin 6A was as effective as nerve growth factor at stimulating neurite outgrowth in PC12 cells, and that its neurotrophic effect was transmitted through signaling by mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinase (PI3K). We further found that neurotrophin-induced neurite formation in PC12 cells could be partially mediated by inhibition of Rif GTPase activity downstream of MAPKs and PI3K signaling. In conclusion, we newly identified Rif as a regulator of the cytoskeletal rearrangement mediated by semaphorins.

The Rif GTPase regulates cytoskeletal signaling from plexinA4 to promote neurite retraction

The small GTPase Rif is required for the early stages of dendritic spine formation in neurons, acting through the formin mDia2 to control actin polymerization. Rif is expressed at high levels in the brain, suggesting broader roles in neuronal function. We screened a yeast two-hybrid cDNA library to identify additional binding partners for Rif of potential relevance to neuronal function. We found that Rif interacts with FARP1, a neuronal activator of the RhoA GTPase. We show that Rif has two separate roles in FARP1 regulation-in controlling its association with plexinA4, and in releasing active RhoA from a plexinA4/FARP1 complex. The regulation of FARP1 by Rif promotes neurite retraction in cells stimulated with the semaphorin Sema6A.

Sulforhodamine B restaining as a whole-cell label allows visualizing one more fluorochrome and its application in assaying protein nucleocytoplasmic distribution

Multiparametric image analysis is highly preferable in revealing complicated biological processes. The multiplexing capability is demanding and is limited by spectral overlap of fluorescent dyes and the number of fluorescent channels available in an imaging platform, especially when both nuclear and cytoplasmic staining are required for the analysis. Here, we introduce a retrospective method that enables cell labeling by restaining the cells with sulforhodamine B (SRB) using a positioning‐stage equipped microscope and an automated image registration technique. Using signal transducers and activator of transcription 1 (STAT1) nuclear translocation as an example, this method was shown to be able to improve the reliability and multiplexing capability of protein distribution assays. The application of this method in a three‐fluorescent channel platform is functionally equal to a conventional four‐color assay and enables the correlation between cellular distributions of two proteins expressed at very low levels to be detected. We have demonstrated this application using STAT3 and syndecan‐1. For the first time, we found that the two proteins were correlated at both the nuclear:cytoplasmic distribution and expression levels. This experimental direction could advance our understanding as to how these molecules function. The simplicity and automation of this method makes it easily applicable into other imaging assays. Other dyes can be used in a similar way as substitutes of SRB.

The synthetic antihyperlipidemic drug potassium piperate selectively kills breast cancer cells through inhibiting G1-S-phase transition and inducing apoptosis

Piper longum L. is a well-known traditional antihyperlipidemic medicine in China, containing medicinal constituents of piperine, pipernonaline and piperlonguminine in its fruit. However, the antitumor properties of these constituents have not yet been studied. We found that potassium piperate (GBK), a derivative of piperine, inhibited proliferation of cancer cells. GBK selectively inhibited the G1-S-phase transition in breast cancer cells and the G1 arrest was correlated with induction of p27 expression, which is an inhibitor for cyclin-dependent kinases, and inhibition of cyclin A, cyclin E and cyclin B expression. Moreover, GBK treatment led to a downregulation of the mini-chromosome maintenance protein expression and induction of mitochondrial-dependent cell apoptosis in breast cancer cells. Our results also suggested that GBK might also inhibit cancer cell proliferation through epigenetic signaling pathways. A synergistic effect in inhibition of cancer cell proliferation was found when GBK was combined with chemotherapy medicines etoposide phosphate or cisplatin at middle or low doses in vitro. These results show that GBK is a novel potential anti-breast cancer drug that inhibits cell proliferation and promotes cell apoptosis.