Cell movements during gastrulation: snail dependent and independent pathways

The morphogenetic process of gastrulation requires multiple inputs and intricate coordination. Genetic analyses demonstrate critical roles of vertebrate and invertebrate Snail proteins in this process. Together with other regulatory molecules including Wnt and BMP, the Snail pathways specify cell fate and reorganize cellular machineries to coordinate morphological changes and cell movements during gastrulation.     

Detection of temporary lateral confinement of membrane proteins using single-particle tracking analysis.

Techniques such as single-particle tracking allow the characterization of the movements of single or very few molecules. Features of the molecular trajectories, such as confined diffusion or directed transport, can reveal interesting biological interactions, but they can also arise from simple Brownian motion. Careful analysis of the data, therefore, is necessary to identify interesting effects from pure random movements. A method was developed to detect temporary confinement in the trajectories of membrane proteins that cannot be accounted for by Brownian motion. This analysis was applied to trajectories of two lipid-linked members of the immunoglobulin superfamily, Thy-1 and a neural cell adhesion molecule (NCAM 125), and the results were compared with those for simulated random walks. Approximately 28% of the trajectories for both proteins exhibited periods of transient confinement, which were < 0.07% likely to arise from random movements. In contrast to these results, only 1.5% of the simulated trajectories showed confined periods. Transient confinement for both proteins lasted on average 8 s in regions that were approximately 280 nm in diameter.     

Developmentally restricted actin-regulatory molecules control morphogenetic cell movements in the zebrafish gastrula

Although our understanding of the regulation of cellular actin and its control during the development of invertebrates is increasing, the question as to how such actin dynamics are regulated differentially across the vertebrate embryo to effect its relatively complex morphogenetic cell movements remains poorly understood. Intercellular signaling that provides spatial and temporal cues to modulate the subcellular localization and activity of actin regulatory molecules represents one important mechanism. Here we explore whether the localized gene expression of specific actin regulatory molecules represents another developmental mechanism. We have identified a cap1 homolog and a novel guanine nucleotide exchange factor (GEF), quattro (quo), that share a restricted gene expression domain in the anterior mesendoderm of the zebrafish gastrula. Each gene is required for specific cellular behaviors during the anterior migration of this tissue; furthermore, cap1 regulates cortical actin distribution specifically in these cells. Finally, although cap1 and quo are autonomously required for the normal behaviors of these cells, they are also nonautonomously required for convergence and extension movements of posterior tissues. Our results provide direct evidence for the deployment of developmentally restricted actin-regulatory molecules in the control of morphogenetic cell movements during vertebrate development.     

Ice shaping properties, similar to that of antifreeze proteins, of a zirconium acetate complex

The control of the growth morphologies of ice crystals is a critical issue in fields as diverse as biomineralization, medicine, biology, civil or food engineering. Such control can be achieved through the ice-shaping properties of specific compounds. The development of synthetic ice-shaping compounds is inspired by the natural occurrence of such properties exhibited by antifreeze proteins. We reveal how a particular zirconium acetate complex is exhibiting ice-shaping properties very similar to that of antifreeze proteins, albeit being a radically different compound. We use these properties as a bioinspired approach to template unique faceted pores in cellular materials. These results suggest that ice-structuring properties are not exclusive to long organic molecules and should broaden the field of investigations and applications of such substances.     

FISH and immunocytochemistry: towards visualising single target molecules in living cells

Knowledge of how molecules interact in space and time is crucial for understanding cellular processes. A host of novel techniques have been developed for the visualisation of single target molecules in living cells, many based on fluorescence in situ hybridisation (FISH) or immunocytochemistry (IC). To extend the applicability of FISH to living cells, special backbone-modified probes and specific conformations (molecular beacons) have been designed. In the case of IC, conventional immunoreagents have been fine-tuned with respect to size and affinity or replaced with new protein scaffolds based on ankyrin repeat proteins. Other key advances include the use of proximity ligation to confirm vicinity binding and the use of quantum dots, which have proven potential for cellular labelling.     

Reaction kinetics in the plasma membrane

A great puzzle in science is establishing a bottom up understanding of life by revealing how a collection of molecules gives rise to a living cell that can survive, communicate, and reproduce. In the confines of physics, chemistry, or material science laboratories where it possible to study complex interactions between molecules in a well-defined environment, our understanding of collective behavior is substantially developed. However, the environment in which molecules of a biological cell perform their functions is far from ideal or controllable. The environment inside cellular regions such as the plasma membrane is heterogeneous and dynamic, and functional molecules such as proteins are both dynamic and promiscuous, as they interact with countless other molecules. This makes it extremely challenging to grasp the inner mechanism of the cells, both experimentally and theoretically. On the bright side, this presents scientists with a colorful playground that waits to be explored: the mesoscopic world inside the cell. This review covers some of the recent experimental and theoretical developments in the study of molecular interactions in the plasma membrane, viewed as a heterogeneous medium where the number of reactants can be small, sometimes countable, and its implications for biological function.     

Locomotive mechanism of Physarum plasmodia based on spatiotemporal analysis of protoplasmic streaming

We investigate how an amoeba mechanically moves its own center of gravity using the model organism Physarum plasmodium. Time-dependent velocity fields of protoplasmic streaming over the whole plasmodia were measured with a particle image velocimetry program developed for this work. Combining these data with measurements of the simultaneous movements of the plasmodia revealed a simple physical mechanism of locomotion. The shuttle streaming of the protoplasm was not truly symmetric due to the peristalsis-like movements of the plasmodium. This asymmetry meant that the transport capacity of the stream was not equal in both directions, and a net forward displacement of the center of gravity resulted. The generality of this as a mechanism for amoeboid locomotion is discussed.     

PAR proteins diffuse freely across the anterior-posterior boundary in polarized C. elegans embryos

Polarization of cells by PAR proteins requires the segregation of antagonistic sets of proteins into two mutually exclusive membrane-associated domains. Understanding how nanometer scale interactions between individual PAR proteins allow spatial organization across cellular length scales requires determining the kinetic properties of PAR proteins and how they are modified in space. We find that PAR-2 and PAR-6, which localize to opposing PAR domains, undergo exchange between well mixed cytoplasmic populations and laterally diffusing membrane-associated states. Domain maintenance does not involve diffusion barriers, lateral sorting, or active transport. Rather, both PAR proteins are free to diffuse between domains, giving rise to a continuous boundary flux because of lateral diffusion of molecules down the concentration gradients that exist across the embryo. Our results suggest that the equalizing effects of lateral diffusion are countered by actin-independent differences in the effective membrane affinities of PAR proteins between the two domains, which likely depend on the ability of each PAR species to locally modulate the membrane affinity of opposing PAR species within its domain. We propose that the stably polarized embryo reflects a dynamic steady state in which molecules undergo continuous diffusion between regions of net association and dissociation.     

Single mRNA molecules demonstrate probabilistic movement in living mammalian cells

Cytoplasmic mRNA movements ultimately determine the spatial distribution of protein synthesis. Although some mRNAs are compartmentalized in cytoplasmic regions, most mRNAs, such as housekeeping mRNAs or the poly-adenylated mRNA population, are believed to be distributed throughout the cytoplasm. The general mechanism by which all mRNAs may move, and how this may be related to localization, is unknown. Here, we report a method to visualize single mRNA molecules in living mammalian cells, and we report that, regardless of any specific cytoplasmic distribution, individual mRNA molecules exhibit rapid and directional movements on microtubules. Importantly, the beta-actin mRNA zipcode increased both the frequency and length of these movements, providing a common mechanistic basis for both localized and nonlocalized mRNAs. Disruption of the cytoskeleton with drugs showed that microtubules and microfilaments are involved in the types of mRNA movements we have observed, which included complete immobility and corralled and nonrestricted diffusion. Individual mRNA molecules switched frequently among these movements, suggesting that mRNAs undergo continuous cycles of anchoring, diffusion, and active transport.     

Tetraspanins: Interactions and interplay with integrins

Tetraspanins are small transmembrane proteins present on the cell surface of almost every eukaryotic cell. Through binding with other transmembrane and intracellular proteins, they regulate diverse cellular processes ranging from cell adhesion and motility to synapse formation and tumor progression. Here, we provide a brief overview of molecular, cellular and clinical studies to illustrate how the multiple functions of this fascinating family of molecules stem from their interplay with multiple molecular partners. In particular, we emphasize the special relationship between tetraspanins and the cell adhesion molecules integrins in regulating cell physiology in health and disease.     

Regulation of kinesin-directed movements

Bidirectional organelle transport along microtubules is most likely mediated by the opposing forces generated by two microtubule-based motors: kinesin and cytoplasmic dynein. Because the direction and timing of organelle movements are controlled by the cell, the activity of one or both of these motor molecules must be regulated. Recent studies demonstrate that kinesin, kinesin-like proteins and kinesin-associated proteins can be phosphorylated, and suggest that changes in their phosphorylation state may modulate kinesin's ability to interact with either microtubules or organelles. Thus, it is possible that phosphorylation regulates kinesin-driven movements.     

Cell-matrix interactions and odontoblast differentiation]

The terminal differentiation of odontoblasts requires the integrity of the cytoskeleton and is controlled by cell-matrix interactions. These interactions implicate both matrix molecules and matrix-associated growth factors. On the one hand, predentin-dentin constituents were found to initiate odontoblast differentiation and to allow the maintenance of this state; TGF-beta or related molecules are implicated. Fibronectin on the other hand can induce the differentiation of second generation odontoblasts and interacts with three high molecular weight proteins present in membrane prepared from dental mesenchymal cells. One of these proteins (165 kDa) was localized on the surface of odontoblasts and is involved in the organization of microfilaments. Two main axes of research will have to be developed in the future in order to understand how matrix molecules and growth factors interactions can be modulated in time and space by epithelial and mesenchymal cells, and how such modulations can affect the phenotype of these cells.     

Switchable fluorophores for protein labeling in living cells

Numerous synthetic fluorophores have been developed that can switch their spectroscopic properties upon interaction with other molecules or by irradiation with light. In recent years, protein-labeling techniques have been introduced that permit the specific attachment of such molecules to proteins of interest in living cells. We review here how the attachment of switchable fluorophores to selected proteins of interest via self-labeling protein tags enables new applications in different areas of biology and discuss how these molecules could be further improved.     

RNA synthetic biology

RNA molecules play important and diverse regulatory roles in the cell by virtue of their interaction with other nucleic acids, proteins and small molecules. Inspired by this natural versatility, researchers have engineered RNA molecules with new biological functions. In the last two years efforts in synthetic biology have produced novel, synthetic RNA components capable of regulating gene expression in vivo largely in bacteria and yeast, setting the stage for scalable and programmable cellular behavior. Immediate challenges for this emerging field include determining how computational and directed-evolution techniques can be implemented to increase the complexity of engineered RNA systems, as well as determining how such systems can be broadly extended to mammalian systems. Further challenges include designing RNA molecules to be sensors of intracellular and environmental stimuli, probes to explore the behavior of biological networks and components of engineered cellular control systems.     

Fluorescent biosensors of protein function

Fluorescent biosensors allow researchers to image and quantify protein activity and small molecule signals in living cells with high spatial and temporal resolution. Genetically encoded sensors are coded by a DNA sequence and hence constructed entirely out of amino acids. These biosensors typically utilize light-emitting proteins, such as derivatives of the green fluorescent protein (GFP), and have been developed for a wide range of small molecules and enzyme activities. Fluorescent biosensors can be genetically targeted to distinct locations within cells, such as organelles and membranes. This feature facilitates elucidation of how protein activities and cellular signals are modulated in different regions of the cell. Improvements in the dynamic range and robustness of sensors have enabled high throughput screening for molecules that act as agonists or antagonists of protein function.     

Integrating an integrin: a direct route to actin

Integrins were so named for their ability to link the extracellular and intracellular skeletons. Now almost 20 years into integrin research, numerous questions remain as to how this interaction is accomplished and how it is modified to achieve a desired phenotype. As the cell adhesion and actin assembly fields are merging in combined approaches, novel actin assembly mechanisms are being uncovered. Some of the earliest identified cytoplasmic linker molecules, believed to mediate integrin-actin binding, are once again the subject of scrutiny as potential dynamic mediators of cell anchorage. It seems plausible that each unique cellular morphology occurs as the result of activation of distinct actin assembly systems that are either stabilized by unique bundling and linker proteins or modified for progression to a new phenotype. While this research initiative is likely to continue rapidly in a forward fashion, it remains to be clarified how integrins assemble the most stable and basic cytoskeletal phenotype, the adherent cell with prominent stress fibers. Recent investigations point towards a shift in the current model of anchoring at the cell periphery by providing both mechanisms and evidence for de novo actin assembly orchestrated by the adhesion site. Lacking a complete pathway from integrin ligation to an integrated extracellular-intracellular skeleton in any single system, this review proposes a simple model of integrin-mediated stress fiber integration by drawing from work in multiple systems.     

Cell membranes: the lipid perspective

Although cell membranes are packed with proteins mingling with lipids, remarkably little is known about how proteins interact with lipids to carry out their function. Novel analytical tools are revealing the astounding diversity of lipids in membranes. The issue is now to understand the cellular functions of this complexity. In this Perspective, we focus on the interface of integral transmembrane proteins and membrane lipids in eukaryotic cells. Clarifying how proteins and lipids interact with each other will be important for unraveling membrane protein structure and function. Progress toward this goal will be promoted by increasing overlap between different fields that have so far operated without much crosstalk.