Magnetic optimization in a multicellular magnetotactic organism

Unicellular magnetotactic prokaryotes, which typically carry a natural remanent magnetic moment equal to the saturation magnetic moment, are the prime example of magnetically optimized organisms. We here report magnetic measurements on a multicellular magnetotactic prokaryote (MMP) consisting of 17 undifferentiated cells (mean from 148 MMPs) with chains of ferrimagnetic particles in each cell. To test if the chain polarities of each cell contribute coherently to the total magnetic moment of the MMP, we used a highly sensitive magnetization measurement technique (1 fAm(2)) that enabled us to determine the degree of magnetic optimization (DMO) of individual MMPs in vivo. We obtained DMO values consistently above 80%. Numerical modeling shows that the probability of reaching a DMO > 80% would be as low as 0.017 for 17 randomly oriented magnetic dipoles. We simulated different scenarios to test whether high DMOs are attainable by aggregation or self-organization of individual magnetic cells. None of the scenarios investigated is likely to yield consistently high DMOs in each generation of MMPs. The observed high DMO values require strong Darwinian selection and a sophisticated reproduction mechanism. We suggest a multicellular life cycle as the most plausible scenario for transmitting the high DMO from one generation to the next.     

Theoretical prerequisites for an experimental corroboration of the existence of a specialized cellular system in control of multicellular organism tissue proliferation

A theoretical analysis of cell proliferation as a selflimiting process designed to maintain the integrity of an entire multicellular organism and based on the principles of a "hypercycle" suggests the need for the existence, starting at a certain level of multicellular organization, of a specialized system in control of tissue proliferation, a system represented by a body of cells capable of both stimulating and inhibiting the proliferation of a variety of cell types. An analysis of experimental data in different fields of the biological science points to certain T-cell populations as probable candidate for the role of cellular regulators of tissue proliferation. Using as an example the induction of murine liver regeneration by the administration of CCL4, the author demonstrates the dynamics of the formation of cells stimulating and inhibiting regeneration, which conforms well to theoretical considerations.     

Nanoscopy in a living multicellular organism expressing GFP

We report superresolution fluorescence microscopy in an intact living organism, namely Caenorhabditis elegans nematodes expressing green fluorescent protein (GFP)-fusion proteins. We also superresolve, by stimulated emission depletion (STED) microscopy, living cultured cells, demonstrating that STED microscopy with GFP can be widely applied. STED with GFP can be performed with both pulsed and continuous-wave lasers spanning a wide wavelength range from at least 556–592 nm. Acquiring subdiffraction resolution images within seconds enables the recording of movies revealing structural dynamics. These results demonstrate that numerous microscopy studies of live samples employing GFP as the marker can be performed at subdiffraction resolution.     

Thinking about Bacillus subtilis as a multicellular organism

Initial attempts to use colony morphogenesis as a tool to investigate bacterial multicellularity were limited by the fact that laboratory strains often have lost many of their developmental properties. Recent advances in elucidating the molecular mechanisms underlying colony morphogenesis have been made possible through the use of undomesticated strains. In particular, Bacillus subtilis has proven to be a remarkable model system to study colony morphogenesis because of its well-characterized developmental features. Genetic screens that analyze mutants defective in colony morphology have led to the discovery of an intricate regulatory network that controls the production of an extracellular matrix. This matrix is essential for the development of complex colony architecture characterized by aerial projections that serve as preferential sites for sporulation. While much progress has been made, the challenge for future studies will be to determine the underlying mechanisms that regulate development such that differentiation occurs in a spatially and temporally organized manner.     

Cell organization and ultrastructure of a magnetotactic multicellular organism

Magnetotactic multicellular aggregates and many-celled magnetotactic prokaryotes have been described as spherical organisms composed of several Gram-negative bacteria capable to align themselves along magnetic fields and swim as a unit. Here we describe a similar organism collected in a large hypersaline lagoon in Brazil. Ultrathin sections and freeze fracture replicas showed that the cells are arranged side by side and face both the external environment and an internal acellular compartment in the center of the organism. This compartment contains a belt of filaments linking the cells, and numerous membrane vesicles. The shape of the cells approaches a pyramid, with the apex pointing to the internal compartment, and the basis facing the external environment. The contact region of two cells is flat and represents the pyramid faces, while the contacts of three or more cells contain cell projections and represent the edges. Freeze-fracture replicas showed a high concentration of intramembrane particles on the edges and also in the region of the outer membrane that faces the external environment. Dark field optical microscopy showed that the whole organism performs a coordinated movement with either straight or helicoidal trajectories. We conclude that the organisms described in this work are, in fact, highly organized prokaryotic multicellular organisms.     

Identification of mammalian protein quality control factors by high-throughput cellular imaging

Protein Quality Control (PQC) pathways are essential to maintain the equilibrium between protein folding and the clearance of misfolded proteins. In order to discover novel human PQC factors, we developed a high-content, high-throughput cell-based assay to assess PQC activity. The assay is based on a fluorescently tagged, temperature sensitive PQC substrate and measures its degradation relative to a temperature insensitive internal control. In a targeted screen of 1591 siRNA genes involved in the Ubiquitin-Proteasome System (UPS) we identified 25 of the 33 genes encoding for 26S proteasome subunits and discovered several novel PQC factors. An unbiased genome-wide siRNA screen revealed the protein translation machinery, and in particular the EIF3 translation initiation complex, as a novel key modulator of misfolded protein stability. These results represent a comprehensive unbiased survey of human PQC components and establish an experimental tool for the discovery of genes that are required for the degradation of misfolded proteins under conditions of proteotoxic stress.     

Regulation of the cytoplasmic quality control protein degradation pathway by BAG2

The cytoplasm is protected against the perils of protein misfolding by two mechanisms: molecular chaperones (which facilitate proper folding) and the ubiquitin-proteasome system, which regulates degradation of misfolded proteins. CHIP (carboxyl terminus of Hsp70-interacting protein) is an Hsp70-associated ubiquitin ligase that participates in this process by ubiquitylating misfolded proteins associated with cytoplasmic chaperones. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. Using a proteomics approach, we have identified BAG2, a previously uncharacterized BAG domain-containing protein, as a common component of CHIP holocomplexes in vivo. Binding assays indicate that BAG2 associates with CHIP as part of a ternary complex with Hsc70, and BAG2 colocalizes with CHIP under both quiescent conditions and after heat shock. In vitro and in vivo ubiquitylation assays indicate that BAG2 is an efficient and specific inhibitor of CHIP-dependent ubiquitin ligase activity. This activity is due, in part, to inhibition of interactions between CHIP and its cognate ubiquitin-conjugating enzyme, UbcH5a, which may in turn be facilitated by ATP-dependent remodeling of the BAG2-Hsc70-CHIP heterocomplex. The association of BAG2 with CHIP provides a cochaperone-dependent regulatory mechanism for preventing unregulated ubiquitylation of misfolded proteins by CHIP.     

Regulation of cellular oncosis by uncoupling protein 2

Cell death can proceed through at least two distinct pathways. Apoptosis is an energy-dependent process characterized morphologically by cell shrinkage, whereas oncosis is a form of cell death induced by energy depletion and initially characterized by cell swelling. We demonstrate in HeLa cells but not in normal diploid fibroblasts that modest increases in the expression level of uncoupling protein 2 (UCP-2) leads to a rapid and dramatic fall in mitochondrial membrane potential and to a reduction of mitochondrial NADH and intracellular ATP. In HeLa cells, increased UCP-2 expression leads to a form of cell death that is not inhibited by the anti-apoptotic gene product Bcl-2 and that morphologically resembles cellular oncosis. We further describe the creation of a dominant interfering mutant of UCP-2 whose expression increases resting mitochondrial membrane potential and selectively increases the resistance to cell death following oncotic but not apoptotic stimuli. These results suggest that distinct genetic programs may regulate the cellular response to either apoptotic or oncotic stimuli.     

Homologous control of protein signaling networks

In a previous paper we introduced a method called augmented sparse reconstruction (ASR) that identifies links among nodes of ordinary differential equation networks, given a small set of observed trajectories with various initial conditions. The main purpose of that technique was to reconstruct intracellular protein signaling networks.In this paper we show that a recursive augmented sparse reconstruction generates artificial networks that are homologous to a large, reference network, in the sense that kinase inhibition of several reactions in the network alters the trajectories of a sizable number of proteins in comparable ways for reference and reconstructed networks. We show this result using a large in-silico model of the epidermal growth factor receptor (EGF-R) driven signaling cascade to generate the data used in the reconstruction algorithm.The most significant consequence of this observed homology is that a nearly optimal combinatorial dosage of kinase inhibitors can be inferred, for many nodes, from the reconstructed network, a result potentially useful for a variety of applications in personalized medicine.     

Building the cellular puzzle: control in multi-level reaction networks

Quantitative conceptual tools dealing with control and regulation of cellular processes have been mostly developed for and applied to the pathways of intermediary metabolism. Yet, cellular processes are organized in different levels, metabolism forming the lowest level in a cascade of processes. Well-known examples are the DNA-mRNA-enzyme-metabolism cascade and the signal transduction cascades consisting of covalent modification cycles. The reaction network that constitutes each level can be viewed as a "module" in which reactions are linked by mass transfer. Although in principle all of these cellular modules are ultimately linked by mass transfer, in practice they can often be regarded as "isolated" from each other in terms of mass transfer. Here modules can interact with each other only by means of regulatory or catalytic effects-a chemical species in one module may affect the rate of a reaction in another module by binding to an enzyme or transport system or by acting as a catalyst. This paper seeks to answer two questions about the control and regulation of such multi-level reaction networks: (i) How can the control properties of the system as a whole be expressed in terms of the control properties of individual modules and the effects between modules? (ii) How do the control properties of a module in its isolated state change when it is embedded in the whole system through its connections with the other modules? In order to answer these questions a quantitative theoretical framework is developed and applied to systems containing two, three or four fully interacting modules; it is shown how it can be extended in principle to n modules. This newly developed theory therefore makes it possible to quantitatively dissect intermodular, internal and external regulation in multi-level systems.     

Network compression as a quality measure for protein interaction networks

With the advent of large-scale protein interaction studies, there is much debate about data quality. Can different noise levels in the measurements be assessed by analyzing network structure? Because proteomic regulation is inherently co-operative, modular and redundant, it is inherently compressible when represented as a network. Here we propose that network compression can be used to compare false positive and false negative noise levels in protein interaction networks. We validate this hypothesis by first confirming the detrimental effect of false positives and false negatives. Second, we show that gold standard networks are more compressible. Third, we show that compressibility correlates with co-expression, co-localization, and shared function. Fourth, we also observe correlation with better protein tagging methods, physiological expression in contrast to over-expression of tagged proteins, and smart pooling approaches for yeast two-hybrid screens. Overall, this new measure is a proxy for both sensitivity and specificity and gives complementary information to standard measures such as average degree and clustering coefficients.     

A new method for manipulating transgenes: engineering heat tolerance in a complex, multicellular organism

BACKGROUND: Heat-shock proteins (hsps) are thought to protect cells against stresses, especially due to elevated temperatures. But while genetic manipulation of hsp gene expression can protect microorganisms and cultured metazoan cells against lethal stress, this has so far not been demonstrated in multicellular organisms. Testing whether expression of an hsp transgene contributes to increased stress tolerance is complicated by a general problem of transgene analysis: if the transgene cannot be targeted to a precise site in the genome, newly observed phenotypes may be due to either the action of the transgene or mutations caused by the transgene insertion. RESULTS: To study the relationship between heat tolerance and hsp expression in Drosophila melanogaster, we have developed a novel method for transgene analysis, based upon the site-specific FLP recombinase. The method employs site-specific sister chromatid exchange to create an allelic series of transgene insertions that share the same integration site, but differ in transgene copy number. Phenotypic differences between members of this series can be confidently attributed to the transgenes. Using such an allelic series and a novel thermotolerance assay for Drosophila embryos, we investigated the role of the 70 kD heat-shock protein, Hsp 70, in thermotolerance. At early embryonic stages, Hsp70 accumulation was rate-limiting for thermotolerance, and elevated Hsp70 expression increased survival at extreme temperatures. CONCLUSION: Our results provide an improved method for analyzing transgenes and demonstrate that, in Drosophila, Hsp70 is a critical thermotolerance factor. They show, moreover, that manipulating the expression of a single hsp can be sufficient to improve the stress tolerance of a complex multicellular organism.     

Molecular chaperones in protein quality control

Proteins must fold into their correct three-dimensional conformation in order to attain their biological function. Conversely, protein aggregation and misfolding are primary contributors to many devastating human diseases, such as prion-mediated infections, Alzheimer's disease, type II diabetes and cystic fibrosis. While the native conformation of a polypeptide is encoded within its primary amino acid sequence and is sufficient for protein folding in vitro, the situation in vivo is more complex. Inside the cell, proteins are synthesized or folded continuously; a process that is greatly assisted by molecular chaperones. Molecular chaperones are a group of structurally diverse and mechanistically distinct proteins that either promote folding or prevent the aggregation of other proteins. With our increasing understanding of the proteome, it is becoming clear that the number of proteins that can be classified as molecular chaperones is increasing steadily. Many of these proteins have novel but essential cellular functions that differ from that of more "conventional" chaperones, such as Hsp70 and the GroE system. This review focuses on the emerging role of molecular chaperones in protein quality control, i.e. the mechanism that rids the cell of misfolded or incompletely synthesized polypeptides that otherwise would interfere with normal cellular function.     

The cell and the organism: the role of subdivisional cell replication in the development and maintenance of the multicellular organism

Evidence of new cell formation from nucleoli (subdivisional cell replication) was observed in vitro and in vivo. Mouse melanoma cells, human fibroblasts, and rat mast cells were observed in tissue culture with phase contrast time-lapse cinematography. Evidence of subdivisional cell replication seen in tissue culture was supported by observations of mast cells, cervical epithelial cells, melanoma cells, keratinocytes, and fungal spores in vivo. Indirect evidence for subdivisional cell replication was the presence of differentiated form and function in nuclei and nucleoli. Synergism between subdivisional replication and mitotic replication (subdivisional expansion) is believed to be a key to morphogenesis, whereby cellular and subdivisional zones act as biologic "molds". It is believed that subdivisional cell replication has a key role in maintenance of differentiated form in multicellular organisms, as well as in morphogenesis.