Evidence that late-endosomal SNARE multimerization complex is promoted by transmembrane segments

Assembly of SNARE proteins into quaternary complexes is a critical step in membrane docking and fusion. Here, we have studied the influence of the transmembrane segments on formation of the late endosomal SNARE complex. The complex was assembled in vitro from full-length recombinant SNAREs and from mutants, where the transmembrane segments were either deleted or replaced by oligo-alanine sequences. We show that endobrevin, syntaxin 7, syntaxin 8, and vti1b readily form a complex. This complex forms a dimer as well as multimeric structures. Interestingly, the natural transmembrane segments accelerate the conversion of the quaternary complex to the dimeric form and are essential for multimerization. These in vitro results suggest that the transmembrane segments are responsible for supramolecular assembly of the endosomal SNARE complex.     

Dazap2 is required for FGF-mediated posterior neural patterning, independent of Wnt and Cdx function

The organization of the embryonic neural plate requires coordination of multiple signal transduction pathways, including fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), and WNTs. Many studies have suggested that a critical component of this process is the patterning of posterior neural tissues by an FGF-caudal signaling cascade. Here, we have identified a novel player, Dazap2, and show that it is required in vivo for posterior neural fate. Loss of Dazap2 in embryos resulted in diminished expression of hoxb9 with a concurrent increase in the anterior marker otx2. Furthermore, we found that Dazap2 is required for FGF dependent posterior patterning; surprisingly, this is independent of Cdx activity. Furthermore, in contrast to FGF activity, Dazap2 induction of hoxb9 is not blocked by loss of canonical Wnt signaling. Functionally, we found that increasing Dazap2 levels alters neural patterning and induces posterior neural markers. This activity overcomes the anteriorizing effects of noggin, and is downstream of FGF receptor activation. Our results strongly suggest that Dazap2 is a novel and essential branch of FGF-induced neural patterning.     

机械阻断神经管的闭合影响爪蟾神经管辐射状切入及神经嵴细胞定向迁移(英文)

神经管闭合缺陷 (NTDs)是一种严重的先天畸形疾病,在新生儿中有千分之一的发病率。神经管融合前后,多种组织参与形态发生运动。神经管一经融合,神经嵴细胞就会向背侧中线方向产生单极突出并向此方向迁移形成神经管的顶部。与此同时,神经管从腹侧开始发生辐射状切入以实现单层化。在此,我们在非洲爪蟾的移植体中机械阻断神经管的闭合以检测其细胞运动及随后的图式形成。结果显示神经管闭合缺陷的移植体不能形成单层化的神经管,并且神经嵴细胞滞留在侧面区域不能向背侧中线迁移,而对神经前体标记基因的检测显示神经管的背腹图式形成并未受到影响。以上结果表明神经管的融合对于辐射状切入和神经嵴细胞向背侧中线方向的迁移过程是必需的,而对于神经管的沿背腹轴方向的图式形成是非必需的。     

一种基于微流控芯片和电化学自组装单层的多细胞表面构图方法

细胞表面构图技术对于研究细胞以及细胞间相互作用具有重要意义。近年来随着微加工技术与表面修饰技术的发展,出现了多种细胞表面构图方法。本研究介绍一种基于微流控芯片和电化学自组装单层修饰的多细胞表面构图方法。该方法通过软光刻技术加工具有2种细胞拦截坝结构阵列的微流控通道来实现2种细胞在芯片表面准确定位;通过表面修饰具有阻碍细胞贴壁特性自组装单层来实现对细胞生长区域的限制;最终实现2种不同细胞近距离表面构图。该方法的优点在于,可以使多种细胞根据需要进行准确的表面构图,细胞区域之间没有物理隔离共享培养微环境,允许不同种细胞通过细胞分泌因子进行相互作用,基底透明表面开放可以使用多种观测手段。该方法可以用于不同种细胞间相互作用的研究。     

Positional Information,Positional Error,and Readout Precision in Morphogenesis: A Mathematical Framework

The concept of positional information is central to our understanding of how cells determine their location in a multicellular structure and thereby their developmental fates. Nevertheless, positional information has neither been defined mathematically nor quantified in a principled way. Here we provide an information-theoretic definition in the context of developmental gene expression patterns and examine the features of expression patterns that affect positional information quantitatively. We connect positional information with the concept of positional error and develop tools to directly measure information and error from experimental data. We illustrate our framework for the case of gap gene expression patterns in the early Drosophila embryo and show how information that is distributed among only four genes is sufficient to determine developmental fates with nearly single-cell resolution. Our approach can be generalized to a variety of different model systems; procedures and examples are discussed in detail.     

Complete predicted three-dimensional structure of the facilitator transmembrane protein and hepatitis C virus receptor CD81: conserved and variable structural domains in the tetraspanin superfamily

Tetraspanins are a superfamily of transmembrane proteins implicated in cellular development, motility, and activation through their interactions with a large range of proteins and with specific membrane microdomains. The complete three-dimensional structure of the tetraspanin CD81 has been predicted by molecular modeling and from the crystallographic structure of the EC2 large extracellular domain. Periodicity of sequence conservation, homology modeling, secondary structure prediction, and protein docking were used. The transmembrane domain appears organized as a four-stranded left-handed coiled coil directly connecting to two helices of the EC2. A smaller extracellular loop EC1 contains a small largely hydrophobic beta-strand that packs in a conserved hydrophobic groove of the EC2. The palmitoylable intracellular N-terminal segment forms an amphipathic membrane-parallel helix. Structural variability occurs mainly in an hypervariable subdomain of the EC2 and in intracellular regions. Therefore, the variable interaction selectivity of tetraspanins originates both from sequence variability within structurally conserved domains and from the occurrence of small structurally variable domains. In CD81 and other tetraspanins, the numerous membrane-exposed aromatic residues are asymmetrically clustered and protrude on one side of the transmembrane domain. This may represent a functional specialization of these two sides for interactions with cholesterol, proteins, or membrane microdomains.     

Analyses of non-leucine-rich repeat (non-LRR) regions intervening between LRRs in proteins

Background

Many proteins have LRR (leucine-rich repeat) units interrupted by non-LRRs which we call IR (non-LRR island region).

Methods

We identified proteins containing LRR@IRs (LRRs having IR) by using a new method and then analyzed their natures and distributions.

Results

LRR@IR proteins were found in over two hundred proteins from prokaryotes and from eukaryotes. These are divided into twenty-one different protein families. The IRs occur one to four times in LRR regions and range in length from 5 to 11,265 residues. The IR lengths in Fungi adenylate cyclases (acys) range from 5 to 116 residues; there are 22 LRR repeats. The IRs in Leishmania proteophosphoglycans (ppgs) vary from 105 to 11,265 residues. These results indicate that the IRs evolved rapidly. A group of LRR@IR proteins—LRRC17, chondroadherin-like protein, ppgs, and four Pseudomonas proteins—have a super motif consisting of an LRR block and its adjacent LRR@IR region. This indicates that the entire super motif experienced duplication. The sequence analysis of IRs offers functional similarity in some LRR@IR protein families.

General significance

This study suggests that various IRs and super motifs provide a great variety of structures and functions for LRRs.     

Conformational change from antiparallel beta-sheet to alpha-helix in a series of depsipeptide, -(Leu-Leu-Lac)(n)-: syntheses, spectroscopic studies, and crystal structures of Boc-Leu-Lac-OEt and Boc-(Leu-Leu-Lac)(n)-OEt (n = 1, 2)

The depsipeptides Boc-Leu-Lac-OEt (1) and Boc-(Leu-Leu-Lac)(n)-OEt (n = 1, 2) (2 and 3, respectively) (Boc = tert-butyloxycarbonyl, Lac = L-lactic acid residue) has been synthesized and studied by crystallographic, CD spectroscopic, and ESI-MS analyses. In the packing cells, those three compounds adopt beta-strand conformations. Each molecule is linked into a dimer (1) or an infinite assembly (2 and 3) by tight hydrogen bonds of the type NH...O==C. Interestingly, the hexamer, 3 shows the first example of antiparallel pleated beta-sheet crystal structure for a depsipeptide molecule. In the packing cells, especially for 3, the ester groups O--C==O are perpendicularly oriented to the amide groups NH--C==O and beta-sheet planes to avoid the interaction between --O--(ester) and O==C. Therefore, when the chain length become longer, the O...O==C repulsion interaction works as a beta-sheet breaker and hence promotes an alpha-helical structure as observed for Boc-(Leu-Leu-Lac)(3)-Leu-Leu-OEt (4) (Oku et al. Biopolymers 2004, 75, 242-254) and Boc-(Leu-Leu-Lac)(n)-OEt (n = 4-6) (5-7) (Katakai et al., Biopolymers 1996, 38, 285-290), in which the O...O==C repulsion does not cause significant structural changes in alpha-helical main chains. Therefore from the structural and spectroscopic analyses, we have found governing factors for the specificity in the beta-sheet and alpha-helix decision in this series of depsipeptides, -(Leu-Leu-Lac)(n)-.