Role of LBPA and Alix in multivesicular liposome formation and endosome organization

What are the components that control the assembly of subcellular organelles in eukaryotic cells? Although membranes can clearly be distorted by cytosolic factors, very little is known about the intrinsic mechanisms that control the biogenesis, shape, and organization of organellar membranes. Here, we found that the unconventional phospholipid lysobisphosphatidic acid (LBPA) could induce the formation of multivesicular liposomes that resembled the multivesicular endosomes that exist where this lipid is found in vivo. This process depended on the same pH gradient that exists across endosome membranes in vivo and was selectively controlled by Alix. In turn, Alix regulated the organization of LBPA-containing endosomes in vivo.

Lessons from tomographic studies of the mammalian Golgi

Basic structure studies of the biosynthetic machinery of the cell by electron microscopy (EM) have underpinned much of our fundamental knowledge in the areas of molecular cell biology and membrane traffic. Driven by our collective desire to understand how changes in the complex and dynamic structure of this enigmatic organelle relate to its pivotal roles in the cell, the comparatively high-resolution glimpses of the Golgi and other compartments of the secretory pathway offered to us through EM have helped to inspire the development and application of some of our most informative, complimentary (molecular, biochemical and genetic) approaches. Even so, no one has yet even come close to relating the basic molecular mechanisms of transport, through and from the Golgi, to its ultrastructure, to everybody's satisfaction. Over the past decade, EM tomography has afforded new insights into structure -function relationships of the Golgi and provoked a re-evaluation of older paradigms. By providing a set of tools for structurally dissecting cells at high-resolution in three-dimensions (3D), EM tomography has emerged as a method for studying molecular cell biology in situ. As we move rapidly toward the establishment of molecular atlases of organelles through advances in proteomics and genomics, tomographic studies of the Golgi offer the tantalizing possibility that one day, we will be able to map the spatio-temporal coordinates of Golgi-related proteins and lipids accurately in the context of 4D cellular space. (c) 2005 Elsevier B.V. All rights reserved.

Capsule and fimbria interaction in Klebsiella pneumoniae

The capsular polysaccharide and type I fimbriae are two of the major surface-located virulence properties associated with the pathogenesis of Klebsiella pneumoniae. The capsule is an elaborate polysaccharide matrix that encases the entire cell surface and provides resistance against many host defense mechanisms. In contrast, type 1 fimbriae are thin adhesive thread-like surface organelles that can extend beyond the capsular matrix and mediate D-mannose-sensitive adhesion to host epithelial cells. These fimbriae are archetypical and consist of a major building block protein (FimA) that comprises the bulk of the organelle and a tip-located adhesin (FimH). It is assumed that the extended major-subunit protein structure permits the FimH adhesin to function independently of the presence of a capsule. In this study, we have employed a defined set of K. pneumoniae capsulated and noncapsulated strains to show that the function of type I fimbriae is actually impeded by the concomitant expression of a polysaccharide capsule. Capsule expression had significant effects on two parameters commonly used to define FimH function, namely, yeast cell agglutination and biofilm formation. Our data suggest that this effect is not due to transcriptional/translational changes in fimbrial gene/protein expression but rather the result of direct physical interference. This was further demonstrated by the fact that we could restore fimbrial function by inhibiting capsule synthesis. It remains to be determined whether the expression of these very different surface components occurs simply via random events of phase variation or in a coordinated manner in response to specific environmental cues.

Caveolin, cholesterol, and lipid bodies

In mammalian cells a complex interplay regulates the distribution of cholesterol between intracellular membrane compartments. One important aspect of cholesterol regulation is intracellular cholesterol storage in neutral lipid storage organelles called lipid droplets or lipid bodies (LBs). Recent work has thrust the LB into the limelight as a complex and dynamic cellular organelle. LBs play a crucial role in maintaining the cellular levels of cholesterol by regulating the interplay between lipid storage, hydrolysis and traffickin,-. Studies of caveolins, caveolar membrane proteins linked to lipid regulation, are providing new insights into the role of LBs in regulating cholesterol balance. (c) 2005 Elsevier Ltd. All rights reserved.

Regulated localization of rab18 to lipid droplets - Effects of lipolytic stimulation and inhibition of lipid droplet catabolism

Rab GTPases are crucial regulators of membrane traffic. Here we have examined a possible association of Rab proteins with lipid droplets (LDs), neutral lipid-containing organelles surrounded by a phospholipid monolayer, also known as lipid bodies, which have been traditionally considered relatively inert storage organelles. Although we found close apposition between LDs and endosomal compartments labeled by expressed Rab5, Rab7, or Rab11 constructs, there was no detectable labeling of the LD surface itself by these Rab proteins. In contrast, GFP-Rab18 localized to LDs and immunoelectron microscopy showed direct association with the monolayer surface. Green fluorescent protein (GFP)-Rab18-labeled LDs underwent oscillatory movements in a localized area as well as sporadic, rapid, saltatory movements both in the periphery of the cell and toward the perinuclear region. In both adipocytes and non-adipocyte cell lines Rab18 localized to a subset of LDs. To gain insights into this specific localization, Rab18 was co-expressed with Cav3(DGV), a truncation mutant of caveolin-3 shown to inhibit the catabolism and motility of lipid droplets. GFP-Rab18 and mRFP-Cav3(DGV) labeled mutually exclusive subpopulations of LDs. Moreover, in 3T3-L1 adipocytes, stimulation of lipolysis increased the localization of Rab18 to LDs, an effect reversed by beta-adrenergic antagonists. These results show that a Rab protein localizes directly to the monolayer surface of LDs. In addition, association with the LD surface was increased following stimulation of lipolysis and inhibited by a caveolin mutant suggesting that recruitment of Rab18 is regulated by the metabolic state of individual LDs.

Antigen-43-mediated autoaggregation impairs motility in Escherichia coli

Functional interaction between bacterial surface-displayed autoaggregation proteins such as antigen 43 (Ag43) of Escherichia coli and motility organelles such as flagella has not previously been described. Here, it has been demonstrated for the first time that Ag43-mediated aggregation can inhibit bacterial motility. Ag43 overexpression produces a dominant aggregation phenotype that overrides motility in the presence of low levels of flagella. In contrast, induction of an increased flagellation state prevents Ag43-mediated aggregation. This phenomenon was observed in naturally occurring subpopulations of E coli as phase variants expressing and not expressing Ag43 revealed contrasting motility phenotypes. The effects were shown to be part of a general mechanism because other short adhesins capable of mediating autoaggregation (AIDA-I and TibA) also impaired motility. These novel insights into the function of bacterial autoaggregation proteins suggest that a balance between these two systems, i.e. autoaggregation and flagellation, influences motility.

Cholesterol-induced caveolin targeting to lipid droplets in adipocytes: A role for caveolar endocytosis

We have investigated the targeting of caveolin to lipid bodies in adipocytes that express high levels of caveolins and contain well-developed lipid droplets. We observed that the lipid droplets isolated from adipocytes of caveolin-1 knock out mice contained dramatically reduced levels of cholesterol, indicating that caveolin is required for maintaining the cholesterol content of this organelle. Analysis of caveolin distribution by cell fractionation and fluorescent light microscopy in 3T3-L1 adipocytes indicated that addition of cholesterol rapidly stimulated translocation of caveolin to lipid droplets. The cholesterol-induced trafficking of caveolins to lipid droplets was shown to be dynamin- and protein kinase C (PKC)-dependent and modulated by src tyrosine kinase activation, suggesting a role for caveolar endocytosis in this novel trafficking pathway. Consistent with this, caveolae budding was stimulated by cholesterol addition. The present data identify lipid droplets as potential target organelles for caveolar endocytosis and demonstrate a role for caveolin-1 in the maintenance of free cholesterol levels in adipocyte lipid droplets.

Lipid droplets: a unified view of a dynamic organelle

Lipid droplets form the main lipid store in eukaryotic cells. Although all cells seem to be able to generate lipid droplets, their biogenesis, regulatory mechanisms and interactions with other organelles remain largely elusive. In this article, we outline some of the recent developments in lipid droplet cell biology. We show the mobile and dynamic nature of this organelle, and advocate the adoption of a unified nomenclature to consolidate terminology in this emerging field.

Regulated Cleavages at the West Nile Virus NS4A-2K-NS4B Junctions Play a Major Role in Rearranging Cytoplasmic Membranes and Golgi Trafficking of the NS4A Protein

A common feature associated with the replication of most RNA viruses is the formation of a unique membrane environment encapsulating the viral replication complex. For their part, flaviviruses are no exception, whereupon infection causes a dramatic rearrangement and induction of unique membrane structures within the cytoplasm of infected cells. These virus-induced membranes, termed paracrystalline arrays, convoluted membranes, and vesicle packets, all appear to have specific functions during replication and are derived from different organelles within the host cell. The aim of this study was to identify which protein(s) specified by the Australian strain of West Nile virus, Kunjin virus (KUNV), are responsible for the dramatic membrane alterations observed during infection. Thus, we have shown using immunolabeling of ultrathin cryosections of transfected cells that expression of the KUNV polyprotein intermediates NS4A-4B and NS213-34A, as well as that of individual NS4A proteins with and without the C-terminal transmembrane domain 2K, resulted in different degrees of rearrangement of cytoplasmic membranes. The formation of the membrane structures characteristic for virus infection required coexpression of an NS4A-NS4B cassette with the viral protease NS2B-3pro which was shown to be essential for the release of the individual NS4A and NS4B proteins. Individual expression of NS4A protein retaining the C-terminal transmembrane domain 2K resulted in the induction of membrane rearrangements most resembling virus-induced structures, while removal of the 2K domain led to a less profound membrane rearrangement but resulted in the redistribution of the NS4A protein to the Golgi apparatus. The results show that cleavage of the KUNV polyprotein NS4A-4B by the viral protease is the key initiation event in the induction of membrane rearrangement and that the NS4A protein intermediate containing the uncleaved C-terminal transmembrane domain plays an essential role in these membrane rearrangements.

Cone photoreceptor oil droplet pigmentation is affected by ambient light intensity

The cone photoreceptors of many vertebrates contain spherical organelles called oil droplets. In birds, turtles, lizards and some lungfish the oil droplets are heavily pigmented and function to filter the spectrum of light incident upon the visual pigment within the outer segment. Pigmented oil droplets are beneficial for colour discrimination in bright light, but at lower light levels the reduction in sensitivity caused by the pigmentation increasingly outweighs the benefits generated by spectral tuning. Consequently, it is expected that species with pigmented oil droplets should modulate the density of pigment in response to ambient light intensity and thereby regulate the amount of light transmitted to the outer segment. In this study, microspectrophotometry was used to measure the absorption spectra of cone oil droplets in chickens (Gallus gallus domesticus) reared under bright (unfiltered) or dim (filtered) sunlight. Oil droplet pigmentation was found to be dependent on the intensity of the ambient light and the duration of exposure to the different lighting treatments. In adult chickens reared in bright light, the oil droplets of all cone types (except the violet-sensitive single cones, whose oil droplet is always non-pigmented) were more densely pigmented than those in chickens reared in dim light. Calculations show that the reduced levels of oil droplet pigmentation in chickens reared in dim light would increase the sensitivity and spectral bandwidth of the outer segment significantly. The density of pigmentation in the oil droplets presumably represents a trade-off between the need for good colour discrimination and absolute sensitivity. This might also explain why nocturnal animals, or those that underwent a nocturnal phase during their evolution, have evolved oil droplets with low pigment densities or no pigmentation or have lost their oil droplets altogether.