Enrique R. Rojas

Chemically mediated mechanical expansion of the pollen tube cell wall.

Morphogenesis of plant cells is tantamount to the shaping of the stiff cell wall that surrounds them. To this end, these cells integrate two concomitant processes: 1), deposition of new material into the existing wall, and 2), mechanical deformation of this material by the turgor pressure. However, due to uncertainty regarding the mechanisms that coordinate these processes, existing models typically adopt a limiting case in which either one or the other dictates morphogenesis. In this report, we formulate a simple mechanism in pollen tubes by which deposition causes turnover of cell wall cross-links, thereby facilitating mechanical deformation. Accordingly, deposition and mechanics are coupled and are both integral aspects of the morphogenetic process. Among the key experimental qualifications of this model are: its ability to precisely reproduce the morphologies of pollen tubes; its prediction of the growth oscillations exhibited by rapidly growing pollen tubes; and its prediction of the observed phase relationships between variables such as wall thickness, cell morphology, and growth rate within oscillatory cells. In short, the model captures the rich phenomenology of pollen tube morphogenesis and has implications for other plant cell types.

Response of Escherichia coli growth rate to osmotic shock

Significance The peptidoglycan cell wall is a universal feature of bacteria that determines their shape, their effect on the human immune system, and their susceptibility to many of our front-line antibiotics. Therefore, it is essential to understand the physiology of this structure. Here, we examine the fundamental biomechanical and biochemical processes that drive cell-wall expansion during cell growth. We demonstrate that, contrary to a long-standing hypothesis, osmotic pressure is not essential for cell-wall expansion of the model bacterium Escherichia coli and that growth of this organism is robust to changes in osmotic pressure. This may be an important adaptation for an enteric bacterium, which regularly faces drastic changes in its osmotic environment during entry and exit from the intestine. It has long been proposed that turgor pressure plays an essential role during bacterial growth by driving mechanical expansion of the cell wall. This hypothesis is based on analogy to plant cells, for which this mechanism has been established, and on experiments in which the growth rate of bacterial cultures was observed to decrease as the osmolarity of the growth medium was increased. To distinguish the effect of turgor pressure from pressure-independent effects that osmolarity might have on cell growth, we monitored the elongation of single Escherichia coli cells while rapidly changing the osmolarity of their media. By plasmolyzing cells, we found that cell-wall elastic strain did not scale with growth rate, suggesting that pressure does not drive cell-wall expansion. Furthermore, in response to hyper- and hypoosmotic shock, E. coli cells resumed their preshock growth rate and relaxed to their steady-state rate after several minutes, demonstrating that osmolarity modulates growth rate slowly, independently of pressure. Oscillatory hyperosmotic shock revealed that although plasmolysis slowed cell elongation, the cells nevertheless “stored” growth such that once turgor was reestablished the cells elongated to the length that they would have attained had they never been plasmolyzed. Finally, MreB dynamics were unaffected by osmotic shock. These results reveal the simple nature of E. coli cell-wall expansion: that the rate of expansion is determined by the rate of peptidoglycan insertion and insertion is not directly dependent on turgor pressure, but that pressure does play a basic role whereby it enables full extension of recently inserted peptidoglycan.

Mechanical crack propagation drives millisecond daughter cell separation in Staphylococcus aureus

Pop goes the coccus Daughter cell separation in Staphylococcus aureus proceeds much like the cracking of an egg. So say Zhou et al., who examined dividing cells with millisecond precision using high-speed videomicroscopy. Rather than proceeding gradually, tiny imperfections in the mother cell wall were seen to crack open, leaving two daughter cells linked by a hinge. Science, this issue p. 574 Daughter cell separation in Staphylococcus aureus proceeds much like the cracking of an egg. When Staphylococcus aureus undergoes cytokinesis, it builds a septum, generating two hemispherical daughters whose cell walls are only connected via a narrow peripheral ring. We found that resolution of this ring occurred within milliseconds (“popping”), without detectable changes in cell volume. The likelihood of popping depended on cell-wall stress, and the separating cells split open asymmetrically, leaving the daughters connected by a hinge. An elastostatic model of the wall indicated high circumferential stress in the peripheral ring before popping. Last, we observed small perforations in the peripheral ring that are likely initial points of mechanical failure. Thus, the ultrafast daughter cell separation in S. aureus appears to be driven by accumulation of stress in the peripheral ring and exhibits hallmarks of mechanical crack propagation.

Mechanical Consequences of Cell-Wall Turnover in the Elongation of a Gram-Positive Bacterium

A common feature of walled organisms is their exposure to osmotic forces that challenge the mechanical integrity of cells while driving elongation. Most bacteria rely on their cell wall to bear osmotic stress and determine cell shape. Wall thickness can vary greatly among species, with Gram-positive bacteria having a thicker wall than Gram-negative bacteria. How wall dimensions and mechanical properties are regulated and how they affect growth have not yet been elucidated. To investigate the regulation of wall thickness in the rod-shaped Gram-positive bacterium Bacillus subtilis, we analyzed exponentially growing cells in different media. Using transmission electron and epifluorescence microscopy, we found that wall thickness and strain were maintained even between media that yielded a threefold change in growth rate. To probe mechanisms of elongation, we developed a biophysical model of the Gram-positive wall that balances the mechanical effects of synthesis of new material and removal of old material through hydrolysis. Our results suggest that cells can vary their growth rate without changing wall thickness or strain by maintaining a constant ratio of synthesis and hydrolysis rates. Our model also indicates that steady growth requires wall turnover on the same timescale as elongation, which can be driven primarily by hydrolysis rather than insertion. This perspective of turnover-driven elongation provides mechanistic insight into previous experiments involving mutants whose growth rate was accelerated by the addition of lysozyme or autolysin. Our approach provides a general framework for deconstructing shape maintenance in cells with thick walls by integrating wall mechanics with the kinetics and regulation of synthesis and turnover.

Strategies for cell shape control in tip-growing cells

PREMISE OF THE STUDYnDespite the large diversity in biological cell morphology, the processes that specify and control cell shape are not yet fully understood. Here we study the shape of tip-growing, walled cells, which have evolved a polar mode of cell morphogenesis leading to characteristic filamentous cell morphologies that extend only apically.nnnMETHODSnWe identified the relevant parameters for the control of cell shape and derived scaling laws based on mass conservation and force balance that connect these parameters to the resulting geometrical phenotypes. These laws provide quantitative testable relations linking morphological phenotypes to the biophysical processes involved in establishing and modulating cell shape in tip-growing, walled cells.nnnKEY RESULTS AND CONCLUSIONSnBy comparing our theoretical results to the observed morphological variation within and across species, we found that tip-growing cells from plant and fungal species share a common strategy to shape the cell, whereas oomycete species have evolved a different mechanism.

Homeostatic Cell Growth Is Accomplished Mechanically through Membrane Tension Inhibition of Cell-Wall Synthesis

Feedback mechanisms are required to coordinate balanced synthesis of subcellular components during cell growth. However, these coordination mechanisms are not apparent at steady state. Here, we elucidate the interdependence of cell growth, membrane tension, and cell-wall synthesis by observing their rapid re-coordination after osmotic shocks in Gram-positive bacteria. Single-cell experiments and mathematical modeling demonstrate that mechanical forces dually regulate cell growth: while turgor pressure produces mechanical stress within the cell wall that promotes its expansion through wall synthesis, membrane tension induces growth arrest by inhibiting wall synthesis. Tension inhibition occurs concurrently with membrane depolarization, and depolarization arrested growth independently of shock, indicating that electrical signals implement the negative feedback characteristic of homeostasis. Thus, competing influences of membrane tension and cell-wall mechanical stress on growth allow cells to rapidly correct for mismatches between membrane and wall synthesis rates, ensuring balanced growth.

Regulation of microbial growth by turgor pressure

Rapid changes in environmental osmolarity are a natural aspect of microbial lifestyles. The change in turgor pressure resulting from an osmotic shock alters the mechanical forces within the cell envelope, and can impact cell growth across a range of timescales, through a variety of mechanical mechanisms. Here, we first summarize measurements of turgor pressure in various organisms. We then review how the combination of microfluidic flow cells and quantitative image analysis has driven discovery of the diverse ways in which turgor pressure mechanically regulates bacterial growth, independent of the effect of cytoplasmic crowding. In Gram-positive, rod-shaped bacteria, reductions in turgor pressure cause decreased growth rate. Moreover, a hypoosmotic shock, which increases turgor pressure and membrane tension, leads to transient inhibition of cell-wall growth via electrical depolarization. By contrast, Gram-negative Escherichia coli is remarkably insensitive to changes in turgor. We discuss the extent to which turgor pressure impacts processes such as cell division that alter cell shape, in particular that turgor facilitates millisecond-scale daughter-cell separation in many Actinobacteria and eukaryotic fission yeast. This diverse set of responses showcases the potential for using osmotic shocks to interrogate how mechanical perturbations affect cellular processes.

The outer membrane is an essential load-bearing element in Gram-negative bacteria

Gram-negative bacteria possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan cell wall and an outer membrane. The envelope is a selective chemical barrier1 that defines cell shape2 and allows the cell to sustain large mechanical loads such as turgor pressure3. It is widely believed that the covalently cross-linked cell wall underpins the mechanical properties of the envelope4,5. Here we show that the stiffness and strength of Escherichia coli cells are largely due to the outer membrane. Compromising the outer membrane, either chemically or genetically, greatly increased deformation of the cell envelope in response to stretching, bending and indentation forces, and induced increased levels of cell lysis upon mechanical perturbation and during L-form proliferation. Both lipopolysaccharides and proteins contributed to the stiffness of the outer membrane. These findings overturn the prevailing dogma that the cell wall is the dominant mechanical element within Gram-negative bacteria, instead demonstrating that the outer membrane can be stiffer than the cell wall, and that mechanical loads are often balanced between these structures.The outer membrane of Gram-negative bacteria is shown to be at least as stiff as the cell wall, and this propertyxa0enables it to protect cells from mechanical pertubations.

A Fresh Look at Growth Oscillations in Pollen Tubes: Kinematic and Mechanistic Descriptions

Pollen tubes exhibit rapid polar growth that can display either stationary (steady) or oscillatory elongation rates. The oscillatory mode of growth provides a window into the network of interactions regulating the morphogenesis of these cells. Empirical studies of oscillatory pollen tube growth have sought to uncover the sequence of cellular events that constitute one oscillatory cycle, while other studies have attempted to formalise the principal causal interactions into computational feedback models. In this chapter, we first review the phenomenon of oscillatory tip growth from a kinematic standpoint. Three key kinematic features have emerged from our analysis: (1) oscillatory cells dominate at high elongation rates, (2) well-defined symmetrical and asymmetrical modes of oscillation are observed, and (3) the oscillation cycle of most pollen tubes unfolds over a fairly well-defined distance, independently of the average elongation rate. We then discuss some mechanistic models aiming to explain oscillatory growth and evaluate their ability to account for the observed kinematic features. Although some of these models have reached a fairly high degree of sophistication, none account for the whole range of kinematic behaviour reported in pollen tubes. We conclude with some suggestions of how current models could be improved.