Autonomy of cell proliferation and developmental programs during Arabidopsis aboveground organ morphogenesis

Elaboration of size and shape in multicellular organisms involves coordinated cell division and cell growth. In higher plants, continuity of cell layer structures exists from the shoot apical meristem (SAM), where organ primordia arise, to mature aboveground organs. To unravel the extent of inter-cell layer coordination during SAM and aboveground organ development, cell division in the epidermis was selectively restricted by expressing two cyclin-dependent kinase inhibitor genes, KRP1/ICK1 and KRP4, driven by the L1 layer-specific AtML1 promoter. The transgenes conferred reduced plant size with striking, distorted lateral organ shape. While epidermal cell division was severely inhibited with compensatory cell size enlargement, the underlying mesophyll/cortex layer kept normal cell numbers and resulted in small, packed cells with disrupted cell files. Our results demonstrate the autonomy of cell number checkpoint in the underlying tissues when epidermal cell division is restricted. Finally, the L1 layer-specific expression of both KRP1/ICK1 and KRP4 showed no effects on the structure and function of the SAM, suggesting that the effects of these cyclin-dependent kinase inhibitors are context dependent.     

Preferential Release of Newly Synthesized Insulin Assessed by a Multi-Label Reporter System Using Pancreatic β-Cell Line MIN6

Newly synthesized hormones have been suggested to be preferentially secreted by various neuroendocrine cells. This observation indicates that there is a distinct population of secretory granules containing new and old hormones. Recent development of fluorescent timer proteins used in bovine adrenal chromaffin cells revealed that secretory vesicles segregate into distinct age-dependent populations. Here, we verify the preferential release of newly synthesized insulin in the pancreatic β-cell line, MIN6, using a combination of multi-labeling reporter systems with both fluorescent and biochemical procedures. This system allows hormones or granules of any age to be labeled, in contrast to the timer proteins, which require fluorescence shift time. Pulse-chase labeling with different color probes distinguishes insulin secretory granules by age, with younger granules having a predominantly intracellular localization rather than at the cell periphery.     

Regulatory mechanisms and function of ERK MAP kinases

Spatiotemporal control of the Ras/ERK MAP kinase signaling pathway is a key factor for determining the specificity of cellular responses including cell proliferation, cell differentiation and cell survival. The fidelity of this signaling is regulated by docking interactions as well as scaffolding. Subcellular localization of ERK is controlled by cytoplasmic ERK anchoring proteins that have a nuclear export signal (NES), such as MEK. In quiescent cells, ERK and MEK localize to the cytoplasm. In response to stimulation, dissociation of the MEK-ERK complex is induced and activated ERK translocates to the nucleus. Recently, several negative regulators for Ras/ERK signaling have been identified and their detailed molecular mechanisms have been analyzed. Among them, Sprouty and Sef act as a temporal and a spatial regulator, respectively, for Ras/ERK signaling. Thus, multiple factors are involved in control of Ras/ERK signaling.     

Isolation and Identification of a Novel β-Carboline Derivative in Salted Radish Roots,Raphanus sativus L.

The methanol extract of salted radish roots contains several precursors of yellow pigment. The main compound was isolated by the use of Toyopearl HW-40S column chromatography, and its structure was determined to be 1-(2′-pyrrolidinethion-3′-yl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid on the basis of an elemental analysis, and IR, UV, FAB-MS and NMR spectroscopy. This compound is presumed to have been the condensation product from the degradation of 4-methylthio-3-butenyl isothiocyanate and l-tryptophan. This carboline compound is considered to play an important role in the formation of the yellow pigment in salted radish roots.     

Arf6 Guanine Nucleotide Exchange Factor Cytohesin-2 Binds to CCDC120 and Is Transported Along Neurites to Mediate Neurite Growth

The mechanism of neurite growth is complicated, involving continuous cytoskeletal rearrangement and vesicular trafficking. Cytohesin-2 is a guanine nucleotide exchange factor for Arf6, an Arf family molecular switch protein, controlling cell morphological changes such as neuritogenesis. Here, we show that cytohesin-2 binds to a protein with a previously unknown function, CCDC120, which contains three coiled-coil domains, and is transported along neurites in differentiating N1E-115 cells. Transfection of the small interfering RNA (siRNA) specific for CCDC120 into cells inhibits neurite growth and Arf6 activation. When neurites start to extend, vesicles containing CCDC120 and cytohesin-2 are transported in an anterograde manner rather than a retrograde one. As neurites continue extension, anterograde vesicle transport decreases. CCDC120 knockdown inhibits cytohesin-2 localization into vesicles containing CCDC120 and diffuses cytohesin-2 in cytoplasmic regions, illustrating that CCDC120 determines cytohesin-2 localization in growing neurites. Reintroduction of the wild type CCDC120 construct into cells transfected with CCDC120 siRNA reverses blunted neurite growth and Arf6 activity, whereas the cytohesin-2-binding CC1 region-deficient CCDC120 construct does not. Thus, cytohesin-2 is transported along neurites by vesicles containing CCDC120, and it mediates neurite growth. These results suggest a mechanism by which guanine nucleotide exchange factor for Arf6 is transported to mediate neurite growth.     

A Src-Tks5 pathway is required for neural crest cell migration during embryonic development

In the adult organism, cell migration is required for physiological processes such as angiogenesis and immune surveillance, as well as pathological events such as tumor metastasis. The adaptor protein and Src substrate Tks5 is necessary for cancer cell migration through extracellular matrix in vitro and tumorigenicity in vivo. However, a role for Tks5 during embryonic development, where cell migration is essential, has not been examined. We used morpholinos to reduce Tks5 expression in zebrafish embryos, and observed developmental defects, most prominently in neural crest-derived tissues such as craniofacial structures and pigmentation. The Tks5 morphant phenotype was rescued by expression of mammalian Tks5, but not by a variant of Tks5 in which the Src phosphorylation sites have been mutated. We further evaluated the role of Tks5 in neural crest cells and neural crest-derived tissues and found that loss of Tks5 impaired their ventral migration. Inhibition of Src family kinases also led to abnormal ventral patterning of neural crest cells and their derivatives. We confirmed that these effects were likely to be cell autonomous by shRNA-mediated knockdown of Tks5 in a murine neural crest stem cell line. Tks5 was required for neural crest cell migration in vitro, and both Src and Tks5 were required for the formation of actin-rich structures with similarity to podosomes. Additionally, we observed that neural crest cells formed Src-Tks5-dependent cell protrusions in 3-D culture conditions and in vivo. These results reveal an important and novel role for the Src-Tks5 pathway in neural crest cell migration during embryonic development. Furthermore, our data suggests that this pathway regulates neural crest cell migration through the generation of actin-rich pro-migratory structures, implying that similar mechanisms are used to control cell migration during embryogenesis and cancer metastasis.