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Cellular Biophysics: Bacterial Endospore, Membranes and Random Fluctuation

2006; Elsevier BV; Volume: 16; Issue: 24 Linguagem: Inglês

10.1016/j.cub.2006.11.010

1879-0445

Vladimir A. Lizunov, Joshua Zimmerberg,

Nanopore and Nanochannel Transport Studies

Purposeful motion of biological processes can be driven by Brownian motion of macromolecular complexes with one-sided binding biasing movement in one direction: a Brownian ratchet, now proposed to explain membrane motion during a phagocytosis-like process in bacteria. Purposeful motion of biological processes can be driven by Brownian motion of macromolecular complexes with one-sided binding biasing movement in one direction: a Brownian ratchet, now proposed to explain membrane motion during a phagocytosis-like process in bacteria. In 1905, Albert Einstein published his papers on Brownian motion and put the last nail in the coffin of arguments against the kinetic-molecular view of matter. Molecules are subject to random fluctuations and undergo eternal motion. He showed how random fluctuations in molecules give rise to seemingly purposeful motion, as in the unidirectional flux that arises from diffusion down a gradient (summarized in [1Einstein A. Investigations on the Theory of the Brownian Movement. Dover, New York1956Google Scholar]). Fifty years later, in 1957, Andew Huxley [2Huxley A.F. Muscle structure and theories of contraction.Prog. Biophys. 1957; 7: 255-317Google Scholar] proposed that thermal fluctuations can give rise to directed motion of biological motors. It was not until the 1990s that theoretical studies by pioneers Fumio Oosawa and George Oster were coupled to biological experimentation by Ron Vale and Sandy Simon, respectively, to develop models involving thermal or Brownian ratchets to explain either mechanisms of mechanochemical transduction of energy or translocation of proteins across membranes [3Vale R.D. Oosawa F. Protein motors and Maxwell's demons: does mechanochemical transduction involve a thermal ratchet?.Adv. Biophys. 1990; 26: 97-134Crossref PubMed Scopus (200) Google Scholar, 4Simon S.M. Peskin C.S. Oster G.F. What drives the translocation of proteins?.Proc. Natl. Acad. Sci. USA. 1992; 89: 3770-3774Crossref PubMed Scopus (366) Google Scholar]. Although the second law of thermodynamics prohibits the production of heat or work from thermal fluctuations, this only holds true in equilibrium. Biological systems are far from thermodynamic equilibrium — equilibrium is synonymous with death, not life. So the gedanken experiments of Maxwell's demon and the Feynman ratchet [5Feynman R.P. Leighton R.B. Sands M. The Feynman Lectures on Physics. vol. 1. Addison-Wesley Publishing, Reading, MA1963Google Scholar] are gaining increasing attention in biology. A deterministic motor moves because of direct forces applied in the direction of movement, while a Brownian motor uses a chemical reaction to prevent backward displacement, thereby rectifying thermal fluctuations into unidirectional motion [6Astumian R.D. Derenyi I. Fluctuation driven transport and models of molecular motors and pumps.Eur. Biophys J. 1998; 27: 474-489Crossref PubMed Scopus (220) Google Scholar]. But fluctuations alone cannot do the work: asymmetry is the hidden energy storage, as for example in a concentration gradient or asymmetric potential profile. Asymmetry, or bias, can either exist prior to any interactions, as in rectifying channels for example, or it can be created during ratcheting, as for example in the distinct ATP-bound versus ADP-bound conformations of motor proteins [7Rousselet J. Salome L. Ajdari A. Prost J. Directional motion of Brownian particles induced by a periodic asymmetric potential.Nature. 1994; 370: 446-448Crossref PubMed Scopus (587) Google Scholar, 8Reimann P. Hanggi P. Introduction to the physics of Brownian motors.Appl. Phys. A. 2002; 75: 169-178Crossref Scopus (307) Google Scholar]. Now, Brownian ratchets have been proposed as the mechanism of membrane motion during endospore formation in bacteria [9Broder D.H. Pogliano K. Forespore engulfment mediated by a ratchet-like mechanism.Cell. 2006; 126: 917-928Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar]. While, to most people, bacterial endospores are associated with the dreaded Bacillus anthracis and Clostridium species tetani, botulinum and perfringens, there is a universe of fascinating processes that occur during sporulation that can shed light on similar eukaryotic processes, such as development, membrane remodeling, phagocytosis and, now, the biophysics of membrane motion. Endospores are dormant cells specialized to allow a bacterium to survive adverse environments for long periods, but to return to the usual 'vegetative' state when favorable conditions are restored. Produced by members of the Firmicute family, they are resistant to many of the agents we use to kill bacteria, such as lysozyme, boiling, drying, radiation and disinfectants, such as alcohol and quarternary ammonium compounds (it is a good thing that Eschericia coli does not make endospores or we would now have to autoclave our spinach!). The first step in the production of an endospore is an asymmetric division of the cytoplasm into two cells: a larger, mother cell and a smaller cell, called the forespore (Figure 1A) [10Errington J. Daniel R.A. Scheffers D.J. Cytokinesis in bacteria.Microbiol. Mol. Biol. Rev. 2003; 67: 52-65Crossref PubMed Scopus (520) Google Scholar, 11Dworkin J. Losick R. Developmental commitment in a bacterium.Cell. 2005; 121: 401-409Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar]. Because, in multicellular organisms, the asymmetric division of the cytoplasm marks the first differentiation step of early development, there has been much interest in this process as a model system [11Dworkin J. Losick R. Developmental commitment in a bacterium.Cell. 2005; 121: 401-409Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar]. Recently, a body of work using fluorescence microscopy of labeled membranes has revealed intriguing aspects of the first stage in endospore formation: engulfment of the newly formed forespore by the mother cell, a phagocytosis-like process in which the forespore becomes wholly enveloped by the mother cell (Figure 1B) [9Broder D.H. Pogliano K. Forespore engulfment mediated by a ratchet-like mechanism.Cell. 2006; 126: 917-928Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 12Abanes-De Mello A. Sun Y.L. Aung S. Pogliano K. A cytoskeleton-like role for the bacterial cell wall during engulfment of the Bacillus subtilis forespore.Genes Dev. 2002; 16: 3253-3264Crossref PubMed Scopus (94) Google Scholar]. The first barrier to engulfment is the septum separating the two cells (Figure 1A). Bacillus subtilis overcomes this problem during sporulation by using specialized proteins that can remove the peptidoglycan wall. Three proteins are required for degradation of the bacterial wall needed for successful engulfment [12Abanes-De Mello A. Sun Y.L. Aung S. Pogliano K. A cytoskeleton-like role for the bacterial cell wall during engulfment of the Bacillus subtilis forespore.Genes Dev. 2002; 16: 3253-3264Crossref PubMed Scopus (94) Google Scholar], and their enzymatic activity suggests a mechanism. The bacterial wall itself is proposed to play the role of a cytoskeleton during engulfment, where the advancement of the membrane is coupled to the hydrolase activity of membrane-anchored proteins. Thus, the peptidoglycan substrate serves as tracks along which the membrane is pulled. Surprisingly, when the cell wall is removed by lysozyme, the engulfment mechanism does not fail: it proceeds even faster, suggesting that the removal of the bacterial wall is the limiting step. Moreover, in the absence of cell wall, the protein machinery described above is no longer needed for engulfment; instead, the engulfment becomes dependent on only two other proteins, SpoIIIAH and SpoIIQ, localized on the mother cell's and forespore's outer membranes, repectively (Figure 1B). The tight binding of these proteins to each other in the space between the membranes suggests that their adhesion is essential for engulfment [9Broder D.H. Pogliano K. Forespore engulfment mediated by a ratchet-like mechanism.Cell. 2006; 126: 917-928Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar]. For the Brownian ratchet of engulfment proposed by Broder and Pogliano [9Broder D.H. Pogliano K. Forespore engulfment mediated by a ratchet-like mechanism.Cell. 2006; 126: 917-928Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar], thermal fluctuations of membrane surface can indeed bring the interacting protein molecules into contact, and the energy of specific bonding used to limit outward fluctuations of the membrane. In this way, the chemical energy of sticky proteins is used to shift the potential energy profile of the system to make interaction of the next pair of sticky molecules more probable. How can this model be tested? We can estimate the fluctuation amplitude, h, when membrane tension, σ, is low (σ≪kBT/h2): then h ∼ [(kBT)2/βP]1/3, where pressure load P = 2σ/a, and a is half of the thickness of the protrusion (from electron microscopy a ∼25 nm [12Abanes-De Mello A. Sun Y.L. Aung S. Pogliano K. A cytoskeleton-like role for the bacterial cell wall during engulfment of the Bacillus subtilis forespore.Genes Dev. 2002; 16: 3253-3264Crossref PubMed Scopus (94) Google Scholar]; see also Figure 2) [13Mogilner A. Oster G. The physics of lamellipodial protrusion.Eur. Biophys. J. 1996; 25: 47-53Crossref Scopus (81) Google Scholar, 14Peskin C.S. Odell G.M. Oster G.F. Cellular motions and thermal fluctuations: the Brownian ratchet.Biophys. J. 1993; 65: 316-324Abstract Full Text PDF PubMed Scopus (723) Google Scholar]. Substituting σ = 0.03 pN/nm, kBT = 4 pN·nm, β = 20 kBT = 80 pN·nm, and a = 25 nm, we calculate that h ∼ 10 nm. The model predicts thresholds for values of σ and β that make fluctuations insufficient to reach the next available protein. Local membrane fluctuations creating high curvature sites can also help to localize specific proteins to the leading edge of the membrane, as suggested for the budding of viral particles [15Lerner D. Deutsch J. Oster G. How does a virus bud?.Biophys. J. 1993; 65: 73-79Abstract Full Text PDF PubMed Scopus (23) Google Scholar]. Figure 3 shows a schematic energy profile for the system of two contacting membranes with sticky molecules.Figure 3Energy profile for the system of two contacting membranes with sticky molecules.Show full captionThe local minimums represent equilibrium states of the membrane, with the smallest bending energy possible under the boundary conditions imposed by the cell wall and anchored sticky molecules. A smaller barrier to the right reflects the minimum fluctuation energy necessary to bring two adjacent sticky molecules into contact. Upon binding, the total energy of the system is lowered by the amount of binding energy, creating the barrier to slipping back. Then the membrane shape relaxes to a new local minimum shifted to the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The local minimums represent equilibrium states of the membrane, with the smallest bending energy possible under the boundary conditions imposed by the cell wall and anchored sticky molecules. A smaller barrier to the right reflects the minimum fluctuation energy necessary to bring two adjacent sticky molecules into contact. Upon binding, the total energy of the system is lowered by the amount of binding energy, creating the barrier to slipping back. Then the membrane shape relaxes to a new local minimum shifted to the right. As Broder and Pogliano [9Broder D.H. Pogliano K. Forespore engulfment mediated by a ratchet-like mechanism.Cell. 2006; 126: 917-928Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar] point out, the success of the engulfment should depend in a threshold-like manner on the amount of proteins. Basically the mean protein–protein distance should be comparable to the amplitude of the membrane fluctuation. We can estimate this minimum number of sticky SpoIIQ proteins on the spore needed for engulfment as follows. A simple square lattice gives us the area per one protein as h2 = 100 nm2, from which we infer that the number of proteins per spore N = πD2/h2 = 3.14(750 nm/10 nm)2 = 1.7 × 104. Other important issues during engulfment are the volume and surface of the mother cell. Topological rearrangements during engulfment inevitably imply changes of surface to volume ratio during engulfment, otherwise pressure would increase membrane tension and dampen fluctuations (decrease their amplitude). There are two extreme cases. In the first scenario, the volume of mother cell is constant during engulfment. Then, the membrane surface area should increase. The mother cell has length L = 3–4 μm and diameter D = 0.8 μm; the spore is round with D = 0.75 μm. Then the surface area of mother cell should increase by πD2 ∼ 1.8 μm2 (∼2 × 106 lipids) or about 25% of its initial value. In the second scenario, the surface area is constant during engulfment. Then, the volume of the mother cell should decrease by roughly the same 25% or πD3/3 ∼ 0.45 μm3 (if D is constant then L should change by 25% or ∼1 μm). In reality, perhaps a combination of both scenarios is present. A small decrease of the volume of the mother cell would decrease tension, increase the amplitude of membrane fluctuations and facilitate membrane invagination on the large scale. On the other side, there could be direct influx of newly synthesized lipid material into the mother cell membrane. Indeed there are indications that de novo lipid biosynthesis is required during engulfment [16Schujman G.E. Grau R. Gramajo H.C. Ornella L. de Mendoza D. De novo fatty acid synthesis is required for establishment of cell type-specific gene transcription during sporulation in Bacillus subtilis.Mol. Microbiol. 1998; 29: 1215-1224Crossref PubMed Scopus (30) Google Scholar]. In both cases engulfment should be sensitive to osmotic effects, and we should look forward to simple tests of these predictions. The 2006 Nobel prize in physics was awarded to John C. Mather and George F. Smoot for testing the idea that fluctuations after the Big Bang were the driving force for creation of the universe, showing us the density fluctuations thought to give rise to formation of galaxies and the present form of the universe. It may be that random thermal fluctuations are also at the heart of the smallest molecular processes within living organisms that appear to us as purposeful unidirectional motion.