Effect of Clathrin Light Chains on the Stiffness of Clathrin Lattices and Membrane Budding

Clathrin‐dependent transport processes require the polymerization of clathrin triskelia into polygonal scaffolds. Together with adapter proteins, clathrin collects cargo and induces membrane bud formation. It is not known to what extent clathrin light chains affect the structural and functional properties of clathrin lattices and the ability of clathrin to deform membranes. To address these issues, we have developed a novel procedure for analyzing clathrin lattice formation on rigid surfaces. We found that lattices can form on adaptor‐coated convex‐, planar‐ and even shallow concave surfaces, but the rate of formation and resistance to thermal dissociation of the lattice are greatly enhanced on convex surfaces. Atomic force microscopy on planar clathrin lattices demonstrates that the stiffness of the clathrin lattice is strictly dependent on light chains. The reduced stiffness of the lattice also compromised the ability of clathrin to generate coated buds on the surface of rigid liposomal membranes.      

CURT1调控类囊体膜弯曲的研究进展

高等植物叶绿体中的基粒是由多个圆盘状类囊体堆叠在一起形成的特殊结构, 它的形成可以将光合蛋白复合体分配在类囊体膜的不同位置, 使类囊体膜具有横向异质性, 能有效进行光合作用。促进基粒形成的关键是使类囊体膜弯曲, 目前发现导致膜弯曲的关键因子是CURT1蛋白。该文对近年在拟南芥(Arabidopsis thaliana)和蓝藻(Cyanobacteria)中有关CURT1蛋白的研究进展进行综述, 并对未来类囊体膜结构与功能的动态调控研究进行展望。     

Curvature-dependent lateral distribution of raft markers in the human erythrocyte membrane

The distribution of raft markers in curved membrane exvaginations and invaginations, induced in human erythrocytes by amphiphile-treatment or increased cytosolic calcium level, was studied by fluorescence microscopy. Cholera toxin subunit B and antibodies were used to detect raft components. Ganglioside GM1 was enriched in membrane exvaginations (spiculae) induced by cytosolic calcium and amphiphiles. Stomatin and the cytosolic proteins synexin and sorcin were enriched in spiculae when induced by cytosolic calcium, but not in spiculae induced by amphiphiles. No enrichment of flotillin-1 was detected in spiculae. Analyses of the relative protein content of released exovesicles were in line with the microscopic observations. In invaginations induced by amphiphiles, the enrichment of ganglioside GM1, but not of the integral membrane proteins flotillin-1 and stomatin, was observed. Based on the experimental results and theoretical considerations we suggest that membrane skeleton-detached, laterally mobile rafts may sort into curved or flat membrane regions dependent on their intrinsic molecular shape and/or direct interactions between the raft elements.     

Lipid-mediated inactivation of colicin E1 channels by calcium ions

Based on the model of a toroidal protein-lipid pore, the effect of calcium ions on colicin E1 channel was predicted. In electrophysiological experiments Ca2+ suppressed the activity of colicin E1 channels in membranes formed of diphytanoylphosphatidylglycerol, whereas no desorption of the protein occurred from the membrane surface. The effect of Ca2+ was not observed on membranes formed of diphytanoylphosphatidylcholine. Single-channel measurements revealed that Ca2+-induced reduction of the colicin-induced current across the negatively charged membrane was due to a decrease in the number of open colicin channels and not changes in their properties. In line with the toroidal model, the effect of Ca2+ on the colicin E1 channel-forming activity is explained by alteration of the membrane lipid curvature caused by electrostatic interaction of Ca2+ with negatively charged lipid head groups.     

Membrane curvature stress and antibacterial activity of lactoferricin derivatives

We have studied correlation of non-lamellar phase formation and antimicrobial activity of two cationic amphipathic peptides, termed VS1-13 and VS1-24 derived from a fragment (LF11) of human lactoferricin on Escherichia coli total lipid extracts. Compared to LF11, VS1-13 exhibits minor, but VS1-24 significantly higher antimicrobial activity. X-ray experiments demonstrated that only VS1-24 decreased the onset of cubic phase formation of dispersions of E. coli lipid extracts, significantly, down to physiological relevant temperatures. Cubic structures were identified to belong to the space groups Pn3m and Im3m. Formation of latter is enhanced in the presence of VS1-24. Additionally, the presence of this peptide caused membrane thinning in the fluid phase, which may promote cubic phase formation. VS1-24 containing a larger hydrophobic volume at the N-terminus than its less active counterpart VS1-13 seems to increase curvature stress in the bilayer and alter the behaviour of the membrane significantly enhancing disruption.     

Specificity of SAF-A and lamin B binding in vitro correlates with the satellite DNA bending state

There is evidence that Matrix Attachment Region (MAR)-binding proteins also bind satellite DNA (satDNA). The aim of the current work was to determine whether the major nuclear matrix (NM) MAR-binding proteins are able to recognize satDNAs of different locations and what DNA structural features are important for the recognition. In nuclei and NM, a number of the same polypeptides were recognized on a southwestern blot when MAR of immunoglobulin kappa gene (Ig kappa MAR) and pericentromeric (periCEN) satDNA fragments were used. However, the binding decreased dramatically when human and mouse CEN satDNA were used for the probes. After an NM extract was subjected to ion exchange chromatography, the main DNA-binding proteins were identified as SAF-A (scaffold attachment factor A) and lamin B. It was not possible to test the binding of lamin B by gel mobility shift assay (GMSA), but SAF-A showed an ability to distinguish CEN and periCEN satDNA fragments in GMSA. While periCEN fragments have an abnormally slow mobility on electrophoresis, which is a hallmark of bent DNA, CEN satDNA fragments have a normal mobility. A computer analysis was done using the wedge model (Ulanovsky and Trifonov [1987] Nature 326:720-722), which describes how the curved state depends on particular nucleotide sequences. The curved states of the fragments predicted by the model are in good agreement with their ability to be recognized by NM proteins. Thus SAF-A and lamin B are able to recognize conserved structural features of satDNA in the same way that MAR-binding proteins recognize MARs in spite of a lack of a consensus sequence. CEN and periCEN satDNAs are distinguished by proteins in correlation with the helical curvature of these fragments.     

Fission of biological membranes: interplay between dynamin and lipids

Membrane budding and fission are the key stages of ubiquitous processes of formation of intracellular transport vesicles. We present a theoretical consideration of one of the most important types of fission machinery, which is mediated by GTPase dynamin and controlled by lipid composition of the membrane. We suggest a mechanism for collapse of a membrane neck driven by interplay between the dynamin collar and the bending elastic energy of the neck membrane. The collar plays a role of a rigid external skeleton, which imposes mechanical constraints on the neck. We show that in certain conditions the membrane of the neck loses its stability and collapses. Collapse can result from: (i) shifting of the spontaneous curvature of the neck membrane towards negative values, (ii) stretching of the dynamin collar, (iii) tightening of the dynamin collar. The three factors can act separately or concertedly. The suggested model accounts for the major experimental knowledge on membrane fission mediated by dynamin. It includes the elements of all previous models of dynamin action based on different sets of experimental results [Sever et al., Traffic 2000; 1: 385-392]. It reconciles, at least partially, the apparent contradictions between the existing alternative views on biomembrane fission machinery.