The role of water in cell architecture

The role of water in biochemical and cellular events is ignored by most workers. However, much recent research has pointed to the importance of physical processes of the cell, which focus attention on such straight forward, elementary questions as position and relationship in space of cell components. In this communication these questions are examined in terms of a new model of water structure. A radically new feature of this model is that water clusters have long-term rather than flickering existence and are as large as the macromolecular components of the cell. These properties allow the clusters and other components to pack together spacially so giving rise to integrated, large-scale, subcellular structures. The intimate participation of water in these structures would explain the fragility of the cytoplasmic organization.     

Stochastic effects in adaptive reconstruction of body damage: implied the creativity of natural selection

After an injury occurs, mechanical/biochemical loads on muscles influence the composition and structure of recovering muscles; this effect likely occurs in other tissues, cells and biological molecules as well owing to the similarity, interassociation and interaction among biochemical reactions and molecules. The ‘damage and reconstruction’ model provides an explanation for how an ideal cytoarchitecture is created by reducing components not suitable for bearing loads; in this model, adaptive changes are induced by promoting the stochasticity of biochemical reactions. Biochemical and mechanical loads can direct the stochasticity of biochemical reactions, which can in turn induce cellular changes. Thus, mechanical and biochemical loads, under natural selection pressure, modify the direction of cell‐ and tissue‐level changes and guide the formation of new structures and traits, thereby influencing microevolution. In summary, the ‘damage and reconstruction’ model accounts for the role of natural selection in the formation of new organisms, helps explain punctuated equilibrium, and illustrates how macroevolution arises from microevolution.     

The lipid bilayer and aquaporins: parallel pathways for water movement into plant cells

The plant plasma membrane is the the major barrier to water flow between cells and their surroundings. Water movement across roots involves pathways comprising many cells and their walls. There are three possible pathways which water can follow, (i) a trans-cellular pathway, which involves serial movement into and out from radial files of cells, (ii) a symplasmic pathway through the plasmodesmata, which creates a cytoplasmic continuum and (iii) a tortuous, extracellular pathway through the cell walls, the apoplasmic pathway. In each of these pathways water movement across cell membranes occurs at some stage. The possible role of water-channels in membranes is discussed in relation to this movement. The molecular identity of water-channel proteins in plasma membranes of plants has been confirmed but there remain a number of unresolved questions about their role in cell and tissue water relations, their interaction with the lipid components of membranes and the relationship between water movement through membranes by diffusion in the bilayer.     

GM1-containing lipid rafts are depleted within clathrin-coated pits

Recent studies show that markers for lipid rafts are among the plasma membrane components most likely to be internalized independently of clathrin-coated pits, and there is evidence to suggest that lipid rafts may play a functional role in endocytic trafficking [1-5]. However, lipid rafts themselves are commonly defined purely in biochemical terms, by resistance to detergent extraction. The existence of rafts in live-cell membranes remains controversial [6-8], and their distribution relative to endocytic machinery has not been investigated. This study employs fluorescence resonance energy transfer (FRET) to show that in the plasma membrane (PM) of living cells the glycosphingolipid GM1, labeled with cholera toxin B subunit (CTB) [9,10], is found at least in part within clusters that also include GPI-linked proteins. These clusters are cholesterol-dependent and exclude non-raft proteins such as transferrin receptor and so possess predicted properties of lipid rafts. This type of lipid raft is largely excluded from clathrin-positive regions of the PM. They are found within Caveolin-positive regions at the same concentration as at the rest of the cell surface. The data provide evidence for a model in which lipid rafts are distributed uniformly across most of the PM of nonpolarized cells but are prevented from entering clathrin-coated pits.     

Oxygen-oxygen distances in protein-bound crystallographic water suggest the presence of protonated clusters

BackgroundThe availability of high-resolution X-ray structures has shown that proteins contain numerous water molecules, but their role is still not fully understood. Protonated and deprotonated water species are often involved in biochemical reactions. However protons are exceedingly difficult to detect directly because they are electron-poor species.MethodsThe oxygen‑oxygen distance of the crystallographic water molecules was analyzed in a large high-resolution data set. Moreover, a detailed analysis was carried out on the protein-bound water in the available structures of carbonic anhydrase II and cytochrome c oxidase, chosen as protein models in which protonated and deprotonated water species play a significant role.ResultsThe analysis shows an excess of water-water distances below the expected value for hydrogen bond. In the cavities and on the surface of the considered model proteins, clusters of water molecules are found, whose structure suggests the presence of chemical species deriving from self-ionization of water.ConclusionsThe presence of a small maximum below the hydrogen bond threshold in the oxygen‑oxygen distance distribution of crystallographic water molecules, along with the location of many of these water clusters, suggest the presence of Zundel-like structures in, or near, the proteins. Particularly significant is the presence of such structures in protein regions which have been identified as proton antennae or channels.General significanceThis work shows the possibilities, still unexplored, offered by this type of analysis in detecting in structures obtained by X-ray diffraction the presence of aqueous protons or hydroxide ions, which are chemical species as important as elusive.     

Signaling clusters in the cell membrane

Large-scale molecular assemblies, or signaling clusters, at the cell membrane are emerging as important regulators of cell signaling. Here, we review new findings and describe shared characteristics common to signaling clusters from a diverse set of cellular systems. The well-known T cell receptor cluster serves as our paradigmatic model. Specifically, each cluster initiates recruitment of hundreds of molecules to the membrane, interacts with the actin cytoskeleton, and contains a significant fraction of the entire signaling process. Probed by recent advancements in patterning and microscopy techniques, the signaling clusters display functional outcomes that are not readily predictable from the individual components.     

Golgi apparatus partitioning during cell division

This review discusses the mitotic segregation of the Golgi apparatus. The results from classical biochemical and morphological studies have suggested that in mammalian cells this organelle remains distinct during mitosis, although highly fragmented through the formation of mitotic Golgi clusters of small tubules and vesicles. Shedding of free Golgi-derived vesicles would consume Golgi clusters and disperse this organelle throughout the cytoplasm. Vesicles could be partitioned in a stochastic and passive way between the two daughter cells and act as a template for the reassembly of this key organelle. This model has recently been modified by results obtained using GFP- or HRP-tagged Golgi resident enzymes, live cell imaging and electron microscopy. Results obtained with these techniques show that the mitotic Golgi clusters are stable entities throughout mitosis that partition in a microtubule spindle-dependent fashion. Furthermore, a newer model proposes that at the onset of mitosis, the Golgi apparatus completely loses its identity and is reabsorbed into the endoplasmic reticulum. This suggests that the partitioning of the Golgi apparatus is entirely dependent on the partitioning of the endoplasmic reticulum. We critically discuss both models and summarize what is known about the molecular mechanisms underlying the Golgi disassembly and reassembly during and after mitosis. We will also review how the study of the Golgi apparatus during mitosis in other organisms can answer current questions and perhaps reveal novel mechanisms.     

Mechanobiology in cortical waves and oscillations

Cortical actin waves have emerged as a widely prevalent phenomena and brought pattern formation to many fields of cell biology. Cortical excitabilities, reminiscent of the electric excitability in neurons, are likely fundamental property of the cell cortex. Although they have been mostly considered to be biochemical in nature, accumulating evidence support the role of mechanics in the pattern formation process. Both pattern formation and mechanobiology approach biological phenomena at the collective level, either by looking at the mesoscale dynamical behavior of molecular networks or by using collective physical properties to characterize biological systems. As such they are very different from the traditional reductionist, bottom-up view of biology, which brings new challenges and potential opportunities. In this essay, we aim to provide our perspectives on what the proposed mechanochemical feedbacks are and open questions regarding their role in cortical excitable and oscillatory dynamics.     

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-beta-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.     

Melanin is an essential component for the integrity of the cell wall of Aspergillus fumigatus conidia

Background  

Aspergillus fumigatus is the most common agent of invasive aspergillosis, a feared complication in severely immunocompromised patients. Despite the recent commercialisation of new antifungal drugs, the prognosis for this infection remains uncertain. Thus, there is a real need to discover new targets for therapy. Particular attention has been paid to the biochemical composition and organisation of the fungal cell wall, because it mediates the host-fungus interplay. Conidia, which are responsible for infections, have melanin as one of the cell wall components. Melanin has been established as an important virulence factor, protecting the fungus against the host's immune defences. We suggested that it might also have an indirect role in virulence, because it is required for correct assembly of the cell wall layers of the conidia.     

Glycogen content during the postnatal differentiation of the Leydig cell in the mouse testis

Summary The concentration and distribution of glycogen in relation to postnatal differentiation of the mouse Leydig cell are studied by biochemical and ultrastructural methods. Glycogen decreases to less than one third in the first twelve days after birth. This decrease is accompanied by modifications of its distribution in the cytoplasm. In the newborn it is abundant and arranged in clusters of beta particles; in the mature Leydig cell, glycogen is found scattered in extremely low concentration interspersed among elements of the endoplasmic reticulum.The role of glycogen during Leydig cell differentiation can be interpreted as a source of energy and/or as a source of building material in the biogenesis of membranous components.This work was supported by Grant M 63,121 from the Population Council, U.S.A.Fellow Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina.     

Centrioles take center stage.

Centrioles are among the most beautiful and mysterious of all cell organelles. Although the ultrastructure of centrioles has been studied in great detail ever since the advent of electron microscopy, these studies raised as many questions as they answered, and for a long time both the function and mode of duplication of centrioles remained controversial. It is now clear that centrioles play an important role in cell division, although cells have backup mechanisms for dividing if centrioles are missing. The recent identification of proteins comprising the different ultrastructural features of centrioles has proven that these are not just figments of the imagination but distinct components of a large and complex protein machine. Finally, genetic and biochemical studies have begun to identify the signals that regulate centriole duplication and coordinate the centriole cycle with the cell cycle.     

In Search of Mitochondrial Mechanisms: Interfield Excursions between Cell Biology and Biochemistry

Developing models of biological mechanisms, such as those involved in respiration in cells, often requires collaborative effort drawing upon techniques developed and information generated in different disciplines. Biochemists in the early decades of the 20th century uncovered all but the most elusive chemical operations involved in cellular respiration, but were unable to align the reaction pathways with particular structures in the cell. During the period 1940–1965 cell biology was emerging as a new discipline and made distinctive contributions to understanding the role of the mitochondrion and its component parts in cellular respiration. In particular, by developing techniques for localizing enzymes or enzyme systems in specific cellular components, cell biologists provided crucial information about the organized structures in which the biochemical reactions occurred. Although the idea that biochemical operations are intimately related to and depend on cell structures was at odds with the then-dominant emphasis on systems of soluble enzymes in biochemistry, a reconceptualization of energetic processes in the 1960s and 1970s made it clear why cell structure was critical to the biochemical account. This paper examines how numerous excursions between biochemistry and cell biology contributed a new understanding of the mechanism of cellular respiration.     

How neuropilin-1 regulates receptor tyrosine kinase signalling: the knowns and known unknowns

Essential roles of NRP1 (neuropilin-1) in cardiovascular development and in neuronal axon targeting during embryogenesis are thought to be mediated primarily through binding of NRP1 to two unrelated types of ligands: the VEGF (vascular endothelial growth factor) family of angiogenic cytokines in the endothelium, and the class 3 semaphorins in neurons. A widely accepted mechanism for the role of NRP1 in the endothelium is VEGF binding to NRP1 and VEGFR2 (VEGF receptor 2) and VEGF-dependent formation of complexes or NRP1-VEGFR2 holoreceptors with enhanced signalling activity and biological function. However, although some basic features of this model are solidly based on biochemical and cellular data, others are open to question. Furthermore, a mechanistic account of NRP1 has to accommodate research which emphasizes the diversity of NRP1 functions in different cell types and particularly an emerging role in signalling by other growth factor ligands for RTKs (receptor tyrosine kinases) such as HGF (hepatocyte growth factor) and PDGF (platelet-derived growth factor). It is uncertain, however, whether the model of NRP1-RTK heterocomplex formation applies in all of these situations. In the light of these developments, the need to explain mechanistically the role of NRP1 in signalling is coming increasingly to the fore. The present article focuses on some of the most important unresolved questions concerning the mechanism(s) through which NRP1 acts, and highlights recent findings which are beginning to generate insights into these questions.     

Cell shape and negative links in regulatory motifs together control spatial information flow in signaling networks

The role of cell size and shape in controlling local intracellular signaling reactions, and how this spatial information originates and is propagated, is not well understood. We have used partial differential equations to model the flow of spatial information from the beta-adrenergic receptor to MAPK1,2 through the cAMP/PKA/B-Raf/MAPK1,2 network in neurons using real geometries. The numerical simulations indicated that cell shape controls the dynamics of local biochemical activity of signal-modulated negative regulators, such as phosphodiesterases and protein phosphatases within regulatory loops to determine the size of microdomains of activated signaling components. The model prediction that negative regulators control the flow of spatial information to downstream components was verified experimentally in rat hippocampal slices. These results suggest a mechanism by which cellular geometry, the presence of regulatory loops with negative regulators, and key reaction rates all together control spatial information transfer and microdomain characteristics within cells.     

A novel regulatory circuit specifies cell fate in the Arabidopsis root epidermis

The specification of distinct cell fates in multicellular organisms is a fundamental process in developmental biology. The Arabidopsis root epidermis, which consists of root-hair cells and non-hair cells, provides a useful model system for studying cell fate specification. In this tissue, the cell fates are determined by their relative position to the underlying cortical cells, and many genes have been identified that regulate this position-dependent cell fate specification. Recent studies using genetic, molecular, and biochemical approaches have shed new light on this process and revealed a complex network of interacting and interdependent components. In particular, a novel regulatory circuit has recently been identified, which includes a lateral inhibition pathway and a feedback loop that enables intercellular communication and ensures that two distinct cell types arise in an appropriate pattern. This regulatory circuit is also influenced by a positional signaling pathway which includes the SCRAMBLED leucine-rich repeat receptor kinase. The studies of cell fate specification in the Arabidopsis root epidermis provide new insights into the molecular strategies used to define distinct cell types in plants.     

Functional architecture of the cell nucleus: towards comprehensive toponome reference maps of apoptosis

We have recently described the MELC/TIS fluorescence robot technology that is capable of colocalizing at least a hundred different molecular cell components in one cell. The technology reveals new hierarchical properties of protein network organisation, referred to as the toponome, in which topologically confined protein clusters are interlocked within the structural framework of the cell. In this study we have applied MELC/TIS to construct a three-dimensional toponome map of the cell nucleus of a single human hepatocyte undergoing apoptosis. The map reveals six different spatially separated toponome domains in the nuclear interior of one apoptotic cell. In the drive to decipher the apoptosis-specific molecular network on the single cell level, the present toponome map is a first milestone towards the construction of much larger maps addressing hundreds of molecular cell components across the stages of apoptosis.