The beta3 chain short arm of laminin-332 (laminin-5) induces matrix assembly and cell adhesion activity of laminin-511 (laminin-10)

The basement membrane (BM) protein laminin-332 (Lm332) (laminin-5) has unique activity and structure as compared with other laminins: it strongly promotes cellular adhesion and migration, and its alpha3, beta3, and gamma2 chains are all truncated in their N-terminal regions (short arms). In the present study, we investigated the biological function of the laminin beta3 chain. When the beta3 chain short arm (beta3SA) was overexpressed in HEK293 cells (beta3SA-HEK), they deposited a large amount of beta3SA and a small amount of laminin-511 (Lm511) (laminin-10) on culture plates. Control HEK293 cells secreted Lm511 but failed to deposit it. The extracellular matrix (ECM) deposited by beta3SA-HEK cells strongly promoted cell attachment and spreading. The beta3SA-HEK ECM did not directly bind Lm511, but it stimulated control HEK293 cells to deposit Lm511 on the culture plates. Although purified beta3SA did not support cell adhesion by itself, it enhanced the cell adhesion activity of Lm511. Experiments with anti-integrin antibodies also suggested that the strong cell adhesion activity of the beta3SA-HEK ECM was derived from the synergistic action of beta3SA and Lm511. It has previously been found that beta3SA binds an unknown cell surface receptor. Taken together, the present study suggests that the short arm of the laminin beta3 chain enhances the matrix assembly of Lm511 and its cell adhesion activity by interacting with its receptor.     

Spatial and temporal gene expression patterns occur during corm development.

We investigated gene expression patterns that occur during taro corm development. Two-dimensional gel electrophoresis identified several different prevalent proteins that accumulate during corm development. Microsequencing studies indicated that some of these proteins are related to taste-modifying proteins, such as curculin and miraculin, and proteins found in other storage organs, such as sporamin and the Kunitz trypsin inhibitor. A curculin-encoding cDNA clone, designated as TC1, was identified that corresponds to a highly prevalent 1-kb corm mRNA. The TC1 mRNA accumulates during corm development, is more prevalent in corm apical than basal regions, and is either absent, or present at low concentrations, in other vegetative organs such as the leaf and root. In situ hybridization experiments showed that the TC1 mRNA is highly concentrated in corm storage parenchyma cells and is absent, or present in reduced concentrations, in other corm cells and tissues. Our results show that corm development is associated with the differentiation of specialized cells and tissues, and that these differentiation events are coupled with the temporal and spatial expression of corm-specific genes.     

The short arm of laminin gamma2 chain of laminin-5 (laminin-332) binds syndecan-1 and regulates cellular adhesion and migration by suppressing phosphorylation of integrin beta4 chain

The proteolytic processing of laminin-5 at the short arm of the gamma2 chain (gamma2sa) is known to convert this laminin from a cell adhesion type to a motility type. Here, we studied this mechanism by analyzing the functions of gamma2sa. In some immortalized or tumorigenic human cell lines, a recombinant gamma2sa, in either soluble or insoluble (coated) form, promoted the adhesion of these cells to the processed laminin-5 (Pr-LN5), and it suppressed their migration stimulated by serum or epidermal growth factor (EGF). Gamma2sa also suppressed EGF-induced tyrosine phosphorylation of integrin beta4 and resultant disruption of hemidesmosome-like structures in keratinocytes. Gamma2sa bound to syndecan-1, and this binding, as well as its cell adhesion activity, was blocked by heparin. By analyzing the activities of three different gamma2sa fragments, the active site of gamma2sa was localized to the NH(2)-terminal EGF-like sequence (domain V or LEa). Suppression of syndecan-1 expression by the RNA interference effectively blocked the activities of domain V capable of promoting cell adhesion and inhibiting the integrin beta4 phosphorylation. These results demonstrate that domain V of the gamma2 chain negatively regulates the integrin beta4 phosphorylation, probably through a syndecan-1-mediated signaling, leading to enhanced cell adhesion and suppressed cell motility.     

Spatial and temporal profiles of growth factor expression during CNS demyelination reveal the dynamics of repair priming

Demyelination is the cause of disability in various neurological disorders. It is therefore crucial to understand the molecular regulation of oligodendrocytes, the myelin forming cells in the CNS. Growth factors are known to be essential for the development and maintenance of oligodendrocytes and are involved in the regulation of glial responses in various pathological conditions. We employed the well established murine cuprizone model of toxic demyelination to analyze the expression of 13 growth factors in the CNS during de- and remyelination. The temporal mRNA expression profile during demyelination and the subsequent remyelination were analyzed separately in the corpus callosum and cerebral cortex using laser microdissection and real-time PCR techniques. During demyelination a similar pattern of growth factor mRNA expression was observed in both areas with a strong up-regulation of NRG1 and GDNF and a slight increase of CNTF in the first week of cuprizone treatment. HGF, FGF-2, LIF, IGF-I, and TGF-ß1 were up-regulated mainly during peak demyelination. In contrast, during remyelination there were regional differences in growth factor mRNA expression levels. GDNF, CNTF, HGF, FGF-2, and BDNF were elevated in the corpus callosum but not in the cortex, suggesting tissue differences in the molecular regulation of remyelination in the white and grey matter. To clarify the cellular source we isolated microglia from the cuprizone lesions. GDNF, IGF-1, and FGF mRNA were detected in the microglial fraction with a temporal pattern corresponding to that from whole tissue PCR. In addition, immunohistochemical analysis revealed IGF-1 protein expression also in the reactive astrocytes. CNTF was located in astrocytes. This study identified seven different temporal expression patterns for growth factors in white and grey matter and demonstrated the importance of early tissue priming and exact orchestration of different steps during callosal and cortical de- and remyelination.     

The fine structure of the melanocytes of the adult mouse hair follicle during their amelanotic phase (telogen and early anagen)

Melanocyte-precursor cells have been described previously in telogen and early anagen hair germs by observation of Epon sections using light microscopy. The existence of these precursor cells has not been generally accepted due to lack of ultrastructural evidence. It is our purpose to describe these cells using transmission electron microscopy. We studied hair germs of the dorsal skin of adult mice in the appropriate stages of the hair cycle prior to melanogenesis. Telogen melanocytes are distinguishable from adjacent keratocytes by their lack of desmosomes, lack of coarse clumps of microfilaments, sparsity of ribosomes, unusually few nuclear pores and presence of dendrites. Melanocytes in early anagen show extensive growth with abundant polysomes, elaborate RER and Golgi zones. They still differ from keratocytes in their lack of desmosomes and clumps of microfilament, and also in the occasional presence of premelanosomes.     

Spatial and temporal expression of actin depolymerizing factors ADF7 and ADF10 during male gametophyte development in Arabidopsis thaliana

The actin cytoskeleton plays a crucial role in many aspects of plant cell development. During male gametophyte development, the actin arrays are conspicuously remodeled both during pollen maturation in the anther and after pollen hydration on the receptive stigma and pollen tube elongation. Remodeling of actin arrays results from the highly orchestrated activities of numerous actin binding proteins (ABPs). A key player in actin remodeling is the actin depolymerizing factor (ADF), which increases actin filament treadmilling rates. We prepared fluorescent protein fusions of two Arabidopsis pollen-specific ADFs, ADF7 and ADF10. We monitored the expression and subcellular localization of these proteins during male gametophyte development, pollen germination and pollen tube growth. ADF7 and ADF10 were differentially expressed with the ADF7 signal appearing in the microspore stage and that of ADF10 only during the polarized microspore stage. ADF7 was associated with the microspore nucleus and the vegetative nucleus of the mature grain during less metabolically active stages, but in germinating pollen grains and elongating pollen tubes, it was associated with the subapical actin fringe. On the other hand, ADF10 was associated with filamentous actin in the developing gametophyte, in particular with the arrays surrounding the apertures of the mature pollen grain. In the shank of elongating pollen tubes, ADF10 was associated with thick actin cables. We propose possible specific functions of these two ADFs based on their differences in expression and localization.     

Spatial and temporal expression of Wnt and Dickkopf genes during murine lens development

Recent studies indicate a role for Wnt signalling in regulating lens cell differentiation (Stump et al., 2003). To further our understanding of this, we investigated the expression patterns of Wnts and Wnt signalling regulators, the Dickkopfs (Dkks), during murine lens development. In situ hybridisation showed that Wnt5a, Wnt5b, Wnt7a, Wnt7b, Wnt8a and Wnt8b genes are expressed throughout the early lens primordia. At embryonic day 14.5 (E14.5), Wnt5a, Wnt5b, Wnt7a, Wnt8a and Wnt8b are reduced in the primary fibres, whereas Wnt7b remains strongly expressed. This trend persists up to E15.5. At later embryonic stages, Wnt expression is predominantly localised to the epithelium and elongating cells at the lens equator. As fibre differentiation progresses, Wnt expression becomes undetectable in the cells of the lens cortex. The one exception is Wnt7b, which continues to be weakly expressed in cortical fibres. This pattern of expression continues through to early postnatal stages. However, by postnatal day 21 (P21), expression of all Wnts is distinctly weaker in the central lens epithelium compared with the equatorial region. This is most notable for Wnt5a, which is barely detectable in the central lens epithelium at P21. Dkk1, Dkk2 and Dkk3 have similar patterns of expression to each other and to the majority of the Wnts during lens development. This study shows that multiple Wnt and Dkk genes are expressed during lens development. Expression is predominantly in the epithelial compartment but is also associated, particularly in the case of Wnt7b, with early events in fibre differentiation.     

Spatial and temporal expression patterns directed by theAgrobacterium tumefaciens T-DNA gene 5 promoter during somatic embryogenesis in carrot

We have analysed the patterns of expression of a gene encoding -glucuronidase (GUS) fused to the promoter of theAgrobacterium tumefaciens T-DNA gene 5 during embryogenesis in carrot,Daucus carota L. Gene expression was monitored by a histochemical assay of -glucuronidase activity. The gene 5 promoter, although of bacterial origin, conferred expression upon the marker gene in all stages of embryo development. The patterns of expression however, differed between embryos in different stages of development. In the globular stage expression was confined to the basal part of the embryo, suggesting that the promoter is sensitive to regulatory functions active in the primary establishment of polarity in the radially symmetric globular embryo. In the heart and torpedo stages of development GUS expression was high in the entire embryonic axis, but not in the cotyledons. During germination expression was reduced in the elongating hypocotyl and radicle, and high levels of expression were detected only in the shoot and root apices. Among the transformed cell lines analysed, one was found that showed an aberrant pattern of GUS expression during embryogenesis, in that expression in the upper part of the embryo was undetectable, and expression was restricted to the root apex in later stages of development. This difference in organ specificity of expression is likely due to a large deletion of the promoter.     

Spatial and temporal expression of an epithelial mucin, Muc-1, during mouse development.

The Muc-1 mucin is found as a transmembrane protein in the apical surface of glandular epithelia. To provide insight into possible functions, we have assessed the timing of expression and the distribution of the Muc-1 protein during mouse embryogenesis using three different techniques: RT-PCR, northern blots and immunohistochemistry. Our results indicate that Muc-1 expression correlates with epithelial differentiation in stomach, pancreas, lung, trachea, kidney and salivary glands. Once started, Muc-1 synthesis continually increases with time, mainly due to epithelial area growth. Our data suggest that expression of the Muc-1 gene is under spatial and temporal control during organogenesis. Although Muc-1 is present in different organs, its expression is not induced systemically, but according to the particular onset of epithelial polarization and branching morphogenesis of each individual organ. It is of particular interest that Muc-1 protein can be detected lining the apical surfaces of the developing lumens when the epithelium of these organs is still undergoing folding and branching, and glandular activity has not yet started. We speculate that Muc-1 may participate in epithelial sheet differentiation/lumen formation during early development of the organs known to express it. This speculation is based on: (1) the detection of Muc-1 expression early during organogenesis, (2) the defined apical localization in different epithelia, (3) the decrease in cell-cell interactions when Muc-1 protein is highly expressed and (4) the possible interaction of its cytoplasmic tail with the actin cytoskeleton. However, it remains to be established using in vitro systems, whether the temporal and local expression of the Muc-1 gene coincident with the morphogenetic events described here is relevant for the process.     

Spatial and temporal control of expression of therapeutic genes using heat shock protein promoters

Heat-shock protein promoters, particularly hsp70, have been used for gene therapy strategies because of their efficiency and the possibility of induction by external heat. This review describes some of the characteristics of hsp70 promoters that make them attractive for use in gene therapy. The human hsp70B promoter is especially promising because of its dose response effect with regard to temperature. Spatial and temporal control of transgene expression using hsp70 promoters necessitates non-invasive methods of local heat deposition and accurate local control of temperature. Special emphasis is given to Focused Ultrasound heating guided by Magnetic Resonance temperature mapping.