Bistability: Requirements on Cell-Volume,Protein Diffusion,and Thermodynamics

Bistability is considered wide-spread among bacteria and eukaryotic cells, useful e.g. for enzyme induction, bet hedging, and epigenetic switching. However, this phenomenon has mostly been described with deterministic dynamic or well-mixed stochastic models. Here, we map known biological bistable systems onto the well-characterized biochemical Schlögl model, using analytical calculations and stochastic spatiotemporal simulations. In addition to network architecture and strong thermodynamic driving away from equilibrium, we show that bistability requires fine-tuning towards small cell volumes (or compartments) and fast protein diffusion (well mixing). Bistability is thus fragile and hence may be restricted to small bacteria and eukaryotic nuclei, with switching triggered by volume changes during the cell cycle. For large volumes, single cells generally loose their ability for bistable switching and instead undergo a first-order phase transition.     

On a heuristic point of view concerning the expression of numerous genes during the cell cycle

The current model of the eukaryotic cell cycle proposes that numerous genes are expressed at different times during the cell cycle. The existence of myriad control points for gene expression leads to theoretical and logical problems for cell cycle control. Each expressed gene requires a control element to appear in a cell-cycle specific manner; this control element requires another control element and so on, ad infinitum. There are also experimental problems with the current model based on ineffective synchronization methods and problems with microarray measurements of mRNA. Equally important, the efficacy of mRNA variation in affecting changes in protein content is negligible. An alternative view of the cell cycle proposes cycle-independent, invariant accumulation of mRNA during the cell cycle with decreases of specific proteins occurring only during the mitotic period of the cell cycle.     

Nuclear movement in filamentous fungi

One of the most striking features of eukaryotic cells is the organization of specific functions into organelles such as nuclei, mitochondria, chloroplasts, the endoplasmic reticulum, vacuoles, peroxisomes or the Golgi apparatus. These membrane-surrounded compartments are not synthesized de novo but are bequeathed to daughter cells during cell division. The successful transmittance of organelles to daughter cells requires the growth, division and separation of these compartments and involves a complex machinery consisting of cytoskeletal components, mechanochemical motor proteins and regulatory factors. Organelles such as nuclei, which are present in most cells in a single copy, must be precisely positioned prior to cytokinesis. In many eukaryotic cells the cleavage plane for cell division is defined by the location of the nucleus prior to mitosis. Nuclear positioning is thus absolutely crucial in the unequal cell divisions that occur during development and embryogenesis. Yeast and filamentous fungi are excellent organisms for the molecular analysis of nuclear migration because of their amenability to a broad variety of powerful analytical methods unavailable in higher eukaryotes. Filamentous fungi are especially attractive models because the longitudinally elongated cells grow by apical tip extension and the organelles are often required to migrate long distances. This review describes nuclear migration in filamentous fungi, the approaches used for and the results of its molecular analysis and the projection of the results to other organisms.     

Dielectric single cell spectra in snow algae

Single cell dielectric spectroscopy is introduced to investigate the passive electric properties of individual snow algal cells at different developmental stages. This non-destructive technique characterises the conductivities and permittivities of cell compartments, such as the cell wall, cytoplasm and membrane. To calculate the conductivities and permittivities in a quantitative manner, multi-shelled models are introduced. Dielectric spectra of snow algae are determined by the cell wall which has been found to have an extremely low permittivity of 3–5. Very likely this is a consequence of the high content of Al-Fe silicates and a low water content. Compared with cells and protoplasts of higher plants, the cell interior has a lower internal conductivity. Received: 20 May 1997 / Accepted: 11 May 1998     

Dynamic remodeling of the endosomal system during formation of Salmonella-induced filaments by intracellular Salmonella enterica

The infection by Salmonella enterica results in the massive remodeling of the endosomal system of eukaryotic host cells. One unique consequence is the formation of long tubular endosomal compartments, so-called Salmonella-induced filaments (SIF). Formation of SIF requires the function of type III secretion system and is a requirement of efficient intracellular proliferation of Salmonella. Using high-resolution live cell imaging approaches and electron microscopy, we report for the first time the highly dynamic characteristics of SIF and their ultrastructural properties. In the early phase of infection (4-5 h), SIF display highly dynamic properties in various types of host cells. SIF extend, branch and contract rapidly, and a stabilized network of SIF is formed later (>or=8 h after infection). The velocities of SIF extension and contraction in the different phases of infection were quantified. Our observations lead to novel models for the modification of host cell transport processes by virulence factors of intracellular Salmonella.     

Evolution of specificity in the eukaryotic endomembrane system

Two hundred years after Darwin's birth, our understanding of genetic mechanisms and cell biology has advanced to a level unimaginable in the 19th century. We now know that eukaryotic cells contain a huge variety of internal compartments, each with their own function, identity and history. For the compartments that together form the membrane-trafficking system, one of the central questions is how that identity is encoded and how it evolved. Here we review the key components involved in membrane-trafficking events, including SNAREs, Rabs, vesicle coats, and tethers and what is known about their evolutionary history. Our current understanding suggests a possible common mechanism by which the membrane-trafficking organelles might have evolved. This model of increased organellar complexity by gene duplication and co-evolution of multiple, interacting, specificity-encoding proteins could well be applicable to other non-endosymbiotic organelles as well. The application of basic evolutionary principles well beyond their original scope has been exceedingly powerful not only in reconstructing the history of cellular compartments, but for medical and applied research as well, and underlines the contributions of Darwin's ideas in modern biology.     

New Organelles by Gene Duplication in a Biophysical Model of Eukaryote Endomembrane Evolution

Extant eukaryotic cells have a dynamic traffic network that consists of diverse membrane-bound organelles exchanging matter via vesicles. This endomembrane system arose and diversified during a period characterized by massive expansions of gene families involved in trafficking after the acquisition of a mitochondrial endosymbiont by a prokaryotic host cell >1.8 billion years ago. Here we investigate the mechanistic link between gene duplication and the emergence of new nonendosymbiotic organelles, using a minimal biophysical model of traffic. Our model incorporates membrane-bound compartments, coat proteins and adaptors that drive vesicles to bud and segregate cargo from source compartments, and SNARE proteins and associated factors that cause vesicles to fuse into specific destination compartments. In simulations, arbitrary numbers of compartments with heterogeneous initial compositions segregate into a few compositionally distinct subsets that we term organelles. The global structure of the traffic system (i.e., the number, composition, and connectivity of organelles) is determined completely by local molecular interactions. On evolutionary timescales, duplication of the budding and fusion machinery followed by loss of cross-interactions leads to the emergence of new organelles, with increased molecular specificity being necessary to maintain larger organellar repertoires. These results clarify potential modes of early eukaryotic evolution as well as more recent eukaryotic diversification.     

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM). Cells are cultivated on transparent slides and incubated with a fluorescent marker homogeneously distributed in their plasma membrane. Illumination occurs by a parallel laser beam under variable angles of total internal reflection (TIR) with different penetration depths of the evanescent electromagnetic field. Recording of fluorescence images upon irradiation at about 10 different angles permits to calculate cell-substrate distances with a precision of a few nanometers. Differences of adhesion between various cell lines, e.g. cancer cells and less malignant cells, are thus determined. In addition, possible changes of cell adhesion upon chemical or photodynamic treatment can be examined. In comparison with other methods of super-resolution microscopy light exposure is kept very small, and no damage of living cells is expected to occur.     

Secretory trafficking in neuronal dendrites

The neuronal secretory pathway represents the intracellular route for proteins involved in synaptic transmission and plasticity, as well as lipids required for outgrowth and remodelling of dendrites and axons. Although neurons use the same secretory compartments as other eukaryotic cells, the enormous distances involved, as well as the unique morphology of the neuron and its signalling requirements, challenge canonical models of secretory pathway organization. Here, we review evidence for a distributed secretory pathway in neurons, suggest mechanisms that may regulate secretory compartment distribution, and discuss the implications of a distributed secretory pathway for neuronal morphogenesis and neural-circuit plasticity.     

Policing Tic 'n' Toc, the doorway to chloroplasts

The organization of eukaryotic cells into different membrane-enclosed compartments requires an ordered and regulated system for targeting and translocating proteins synthesized in the cytosol across organellar membranes. Protein translocation through integral membrane proteinaceous complexes shares common principles in different organelles, whereas molecular mechanisms and energy requirements are diverse. Translocation into mitochondria and plastids requires most proteins to cross two membranes, and translocation must be regulated to accommodate environmental or metabolic changes. In the last decade, the first ideas were formulated about the regulation of protein translocation into chloroplasts, thereby laying the foundation for this field. Here, we describe recent models for the regulation of translocation by precursor protein phosphorylation, receptor dimerization, redox sensing and calcium signaling. We suggest how these mechanisms might fit within the regulatory framework for the entry of proteins into chloroplasts.     

Golgi organization and the apical extension of fungal hyphae: an essential relationship

The Golgi apparatus performs crucial functions in the sorting and processing of proteins destined for secretion from eukaryotic cells. In filamentous fungi, organization of the Golgi apparatus reflects the unique challenges brought about by the highly polarized nature of hyphal growth. Recent results show that Golgi compartments are spatially segregated within hyphal tip cells in a manner that depends upon the integrity of the cytoskeleton. Moreover, loss of normal Golgi organization stops polarized hyphal extension and triggers de‐polarization of the hyphal tip. These results emphasize the point that a spatially organized and dynamic Golgi apparatus represents an adaptation that is as important for hyphal extension as is the presence of a Spitzenkörper. In addition, they also identify regulatory mechanisms that could enable controlled de‐polarization of hyphae during development or infection‐related morphogenesis.     

Analysis of Cell Flow and Cell Loss Following X-Irradiation Using Sequential Investigation of the Total Number of Cells In the Various Parts of the Cell Cycle

The cell flow and cell loss of an in vivo growing Ehrlich ascites tumour were calculated by sequential estimation of changes in the total number of cells in the cell cycle compartments. Normal growth was compared with the grossly disturbed cell flow evident after a 5 Gy X-irradiation. The doubling time of normal, exponentially growing cells was 24 hr. the generation time was 21 hr based on double-isotope labelling studies and the potential doubling time was 21 hr. Thus, the growth fraction was 1.0 and the cell loss rate about 0.5%/hr. Following irradiation, a transiently increased relative outflow rate from all cell cycle compartments was found at about 3 and 40 hr, and from S phase at 24 hr after irradiation. Minimum flow rates from all compartments were found up to 20 hr. Cell loss as calculated from the cell flow was compared with non-viable cells determined by Percoll density separation. Increase in cell loss as well as non-viable cells was observed at 24 hr after irradiation at the time of release of the irradiation-induced G₂ blockage. Up to 50 hr, about 70% of the initial total number of cells were lost. the experiments show the applicability and limitations of cell flow and cell loss calculations by sequential analysis of the total number of cells in the various parts of the cell cycle.     

Quantitative elucidation of a distinct spatial gradient-sensing mechanism in fibroblasts

Migration of eukaryotic cells toward a chemoattractant often relies on their ability to distinguish receptor-mediated signaling at different subcellular locations, a phenomenon known as spatial sensing. A prominent example that is seen during wound healing is fibroblast migration in platelet-derived growth factor (PDGF) gradients. As in the well-characterized chemotactic cells Dictyostelium discoideum and neutrophils, signaling to the cytoskeleton via the phosphoinositide 3-kinase pathway in fibroblasts is spatially polarized by a PDGF gradient; however, the sensitivity of this process and how it is regulated are unknown. Through a quantitative analysis of mathematical models and live cell total internal reflection fluorescence microscopy experiments, we demonstrate that PDGF detection is governed by mechanisms that are fundamentally different from those in D. discoideum and neutrophils. Robust PDGF sensing requires steeper gradients and a much narrower range of absolute chemoattractant concentration, which is consistent with a simpler system lacking the feedback loops that yield signal amplification and adaptation in amoeboid cells.     

Dual compartmental localization and function of mammalian NADP+-specific isocitrate dehydrogenase in yeast

Isozymes of NADP⁺-specific isocitrate dehydrogenase (IDP) provide NADPH in cytosolic, mitochondrial, and peroxisomal compartments of eukaryotic cells. Analyses of purified IDP isozymes from yeast and from mouse suggest a general correspondence of pH optima for catalysis and pI values with pH values reported for resident cellular compartments. However, mouse IDP2, which partitions between cytosolic and peroxisomal compartments in mammalian cells, exhibits a broad pH optimum and an intermediate pI value. Mouse IDP2 was found to similarly colocalize in both cellular compartments when expressed in yeast at levels equivalent to those of endogenous yeast isozymes. The mouse enzyme can compensate for loss of yeast cytosolic IDP2 and of peroxisomal IDP3. Removal of the peroxisomal targeting signal of the mouse enzyme precludes both localization in peroxisomes and compensation for loss of yeast IDP3.     

THE CELL POPULATION KINETICS OF NEUTROPHILIC CELLS

Autoradiographic data for the entry of tritiated thymidine labelled cells into the post-proliferative neutrophilic cell compartments following a single injection of isotope have been analysed in terms of two cell kinetic models which differ in the assumed relationships between cell maturation and division. Comparisons with the experimental data were made in an attempt to assess the validities of the models, and kinetic parameters for the compartments of recognizable neutrophilic cells were estimated. Control mechanisms which have been proposed for the granulocyte system are discussed in terms of the kinetic models which were chosen in their determination. Although it was not possible to make a clear choice between the proposed models, preference was established for a random model which did not involve cell loss.     

Charge screening by internal pH and polyvalent cations as a mechanism for activation, inhibition, and rundown of TRPM7/MIC channels

The Mg2+-inhibited cation (MIC) current, believed to represent activity of TRPM7 channels, is found in lymphocytes and mast cells, cardiac and smooth muscle, and several other eukaryotic cell types. MIC current is activated during whole-cell dialysis with divalent-free internal solutions. Millimolar concentrations of intracellular Mg2+ (or other divalent metal cations) inhibit the channels in a voltage-independent manner. The nature of divalent inhibition and the mechanism of channel activation in an intact cell remain unknown. We show that the polyamines (spermine, spermidine, and putrescine) inhibit the MIC current, also in a voltage-independent manner, with a potency that parallels the number of charges. Neomycin and poly-lysine also potently inhibited MIC current in the absence of Mg2+. These same positively charged ions inhibited IRK1 current in parallel with MIC current, suggesting that they probably act by screening the head group phosphates on PIP2 and other membrane phospholipids. In agreement with this hypothesis, internal protons also inhibited MIC current. By contrast, tetramethylammonium, tetraethylammonium, and hexamethonium produced voltage-dependent block but no inhibition. We show that inhibition by internal polyvalent cations can be relieved by alkalinizing the cytosol using externally applied ammonium or by increasing pH in inside-out patches. Furthermore, in perforated-patch and cell-attached recordings, when intracellular Mg2+ is not depleted, endogenous MIC or recombinant TRPM7 currents are activated by cytosolic alkalinization and inhibited by acidification; and they can be reactivated by PIP2 following rundown in inside-out patches. We propose that MIC (TRPM7) channels are regulated by a charge screening mechanism and may function as sensors of intracellular pH.