Hierarchical transitions and fractal wrinkling drive bacterial pellicle morphogenesis

Bacterial cells can self-organize into structured communities at fluid–fluid interfaces. These soft, living materials composed of cells and extracellular matrix are called pellicles. Cells residing in pellicles garner group-level survival advantages such as increased antibiotic resistance. The dynamics of pellicle formation and, more generally, how complex morphologies arise from active biomaterials confined at interfaces are not well understood. Here, using Vibrio cholerae as our model organism, a custom-built adaptive stereo microscope, fluorescence imaging, mechanical theory, and simulations, we report a fractal wrinkling morphogenesis program that differs radically from the well-known coalescence of wrinkles into folds that occurs in passive thin films at fluid–fluid interfaces. Four stages occur: growth of founding colonies, onset of primary wrinkles, development of secondary curved ridge instabilities, and finally the emergence of a cascade of finer structures with fractal-like scaling in wavelength. The time evolution of pellicle formation depends on the initial heterogeneity of the film microstructure. Changing the starting bacterial seeding density produces three variations in the sequence of morphogenic stages, which we term the bypass, crystalline, and incomplete modes. Despite these global architectural transitions, individual microcolonies remain spatially segregated, and thus, the community maintains spatial and genetic heterogeneity. Our results suggest that the memory of the original microstructure is critical in setting the morphogenic dynamics of a pellicle as an active biomaterial.

Spontaneous folding, wrinkling, and curling of soft tissues and active materials are ubiquitous in nature. For example, during the development of mammalian organs and plant leaves, collections of many cells self-organize into ordered morphological structures far larger than the size of the individual cells. Nontrivial geometric patterns and architectures often link form to function, such as the fractal branching and size scaling of leaf veins (1), the gyrification of the cerebral cortex (2–4), and the curving of villi in the gut (5, 6). Bacterial cells can also self-organize into communities known as biofilms and pellicles at interfaces. These multicellular communities are crucial in contexts such as medical infections and industrial biofouling because cells in biofilms and pellicles display enhanced resilience to antibiotics, immune clearance, and physical perturbations compared to their isogenic planktonic counterparts (7–9). Analogous to eukaryotic systems, bacterial biofilms and pellicles develop striking macroscopic morphologies including wrinkles and delaminations that are driven by combined biological, material-physics, and mechanical determinants (10–14). Studies of biofilms at the single-cell level show the emergence of internal cell ordering (15–17) and collective cellular flow (18). However, the dynamics of self-organization and the sequence of mechanical instabilities that direct morphogenesis for expanding active soft materials at fluid–fluid interfaces, such as bacterial pellicles, are undefined. Critically, overarching principles connecting microscopic cell organization to macroscopic structures, while potentially common to morphogenesis across different biological systems, are not well understood.Here, we report the sequence of architectural transitions that occur during pellicle maturation for the pathogen Vibrio cholerae. We discover a morphogenic transition characterized by a cascade of wrinkles with fractal scaling in wavelength, which is distinct from the classic wrinkle-to-fold transition widely observed in physical systems such as thin polymer films on fluid baths (19), nanoparticle thin sheets (20), and thin epitaxial layers of gold and elastomers (21, 22). In classic wrinkle-to-fold transitions, as compression increases beyond the linear regime, the initial wrinkles coalesce and localize into folds with large amplitudes. During such transitions, the shape evolution of passive films can be characterized by the minimization of the system’s total energy for different conformations (20, 23–25). In particular, elastic bending energies contained in small wavelength undulations are lost in favor of energies associated with large-scale folds. By contrast, as we demonstrate below, the opposite length-scale progression occurs for actively growing V. cholerae pellicles, in which finer wrinkles and creases continue to emerge following initial morphogenic transitions. The length scales of wrinkles in a mature V. cholerae pellicle follow fractal order, achieving a pattern with fractal dimension of ∼1.6. The additional dimensionality conferred by the fractal geometry could promote nutrient access and enhance signal transduction compared to a smooth structure (26). The fractal progression toward small length scales, as opposed to coalescence toward larger folds, stems from heterogeneous growth in the initial film. We find that the original distribution of microcolonies is preserved during pellicle architectural transitions, and, importantly, it determines the exact sequence of morphogenesis events that will occur. Indeed, changing the initial colony seeding density enabled us to identify a total of four morphogenic routes, which we termed the standard, bypass, crystalline, and incomplete modes. Our results demonstrate a direct connection between microscopic structure and macroscopic morphology for an active, growing soft biomaterial.     

Transforming growth factor‐beta 3(Tgf‐β3) in a collagen gel delays fusion of the rat posterior interfrontal suture in vivo

Postnatal expansion of the intramembranous bones of the craniofacial skeleton occurs as bone growth at sutures. Loss of the bone growth site occurs when the suture fails to form, or when the newly formed sutures become ossified, resulting in premature obliteration. Previous experiments demonstrated that removal of dura mater from fetal rat coronal sutures, or neutralizing transforming growth factor‐beta 2 (Tgf‐β2) activity using antibodies resulted in premature obliteration of the suture in vitro. Conversely, addition of Tgf‐β3 to coronal sutures in vitro rescued them from osseous obliteration. To examine whether Tgf‐β3 rescues sutures from obliteration in vivo, a collagen gel was used as a vehicle to deliver Tgf‐β3 to the normally fusing rat posterior interfrontal (IF) suture. Surgery was done on postnatal day 9 (P9) rats, in which collagen gels containing 0, 3, or 30 ng Tgf‐β3 were placed above the IF suture, underneath the periosteum for 2 weeks. By P24, 75–100% of animals in control unoperated, sham‐operated, and collagen gel‐only groups had fused IF sutures. In contrast, 40% of sutures exposed to 3 ng Tgf‐β3 remained open, while sutures exposed to 30 ng Tgf‐β were similar to controls. By immunohistochemistry, sutures rescued from obliteration by Tgf‐β3 had the same Tgf‐β receptor type II (Tβr‐II) distribution as controls. However, Tgf‐β3‐treated sutures had altered Tgf‐β2 and Tβr‐I distribution compared to controls. Anat Rec 267:120–130, 2002. © 2002 Wiley‐Liss, Inc.     

Universal rule for the symmetric division of plant cells

The division of eukaryotic cells involves the assembly of complex cytoskeletal structures to exert the forces required for chromosome segregation and cytokinesis. In plants, empirical evidence suggests that tensional forces within the cytoskeleton cause cells to divide along the plane that minimizes the surface area of the cell plate (Errera's rule) while creating daughter cells of equal size. However, exceptions to Errera's rule cast doubt on whether a broadly applicable rule can be formulated for plant cell division. Here, we show that the selection of the plane of division involves a competition between alternative configurations whose geometries represent local area minima. We find that the probability of observing a particular division configuration increases inversely with its relative area according to an exponential probability distribution known as the Gibbs measure. Moreover, a comparison across land plants and their most recent algal ancestors confirms that the probability distribution is widely conserved and independent of cell shape and size. Using a maximum entropy formulation, we show that this empirical division rule is predicted by the dynamics of the tense cytoskeletal elements that lead to the positioning of the preprophase band. Based on the fact that the division plane is selected from the sole interaction of the cytoskeleton with cell shape, we posit that the new rule represents the default mechanism for plant cell division when internal or external cues are absent.     

Predataxis behavior in Myxococcus xanthus

Spatial organization of cells is important for both multicellular development and tactic responses to a changing environment. We find that the social bacterium, Myxococcus xanthus utilizes a chemotaxis (Che)-like pathway to regulate multicellular rippling during predation of other microbial species. Tracking of GFP-labeled cells indicates directed movement of M. xanthus cells during the formation of rippling wave structures. Quantitative analysis of rippling indicates that ripple wavelength is adaptable and dependent on prey cell availability. Methylation of the receptor, FrzCD is required for this adaptation: a frzF methyltransferase mutant is unable to construct ripples, whereas a frzG methylesterase mutant forms numerous, tightly packed ripples. Both the frzF and frzG mutant strains are defective in directing cell movement through prey colonies. These data indicate that the transition to an organized multicellular state during predation in M. xanthus relies on the tactic behavior of individual cells, mediated by a Che-like signal transduction pathway.     

Unveiling the structural basis for translational ambiguity tolerance in a human fungal pathogen

In a restricted group of opportunistic fungal pathogens the universal leucine CUG codon is translated both as serine (97%) and leucine (3%), challenging the concept that translational ambiguity has a negative impact in living organisms. To elucidate the molecular mechanisms underlying the in vivo tolerance to a nonconserved genetic code alteration, we have undertaken an extensive structural analysis of proteins containing CUG-encoded residues and solved the crystal structures of the two natural isoforms of Candida albicans seryl-tRNA synthetase. We show that codon reassignment resulted in a nonrandom genome-wide CUG redistribution tailored to minimize protein misfolding events induced by the large-scale leucine-to-serine replacement within the CTG clade. Leucine or serine incorporation at the CUG position in C. albicans seryl-tRNA synthetase induces only local structural changes and, although both isoforms display tRNA serylation activity, the leucine-containing isoform is more active. Similarly, codon ambiguity is predicted to shape the function of C. albicans proteins containing CUG-encoded residues in functionally relevant positions, some of which have a key role in signaling cascades associated with morphological changes and pathogenesis. This study provides a first detailed analysis on natural reassignment of codon identity, unveiling a highly dynamic evolutionary pattern of thousands of fungal CUG codons to confer an optimized balance between protein structural robustness and functional plasticity.     

Sublimation-driven morphogenesis of Zen stones on ice surfaces

In this article, the formation of Zen stones on frozen lakes and the shape of the resulting pedestal are elucidated. Zen stones are natural structures in which a stone, initially resting on an ice surface, ends up balanced atop a narrow ice pedestal. We provide a physical explanation for their formation, sometimes believed to be caused by the melting of the ice. Instead, we show that slow surface sublimation is indeed the physical mechanism responsible for the differential ablation. Far from the stone, the sublimation rate is governed by the diffuse sunlight, while in its vicinity, the shade it creates inhibits the sublimation process. We reproduced the phenomenon in laboratory-scale experiments conducted in a lyophilizer and studied the dynamics of the morphogenesis. In this apparatus, which imposes controlled constant sublimation rate, a variety of model stones consisting of metal disks was used, which allows us to rule out the possible influence of the thermal conduction in the morphogenesis process. Instead, we show that the stone only acts as an umbrella whose shade hinders the sublimation, hence protecting the ice underneath, which leads to the formation of the pedestal. Numerical simulations, in which the local ablation rate of the surface depends solely on the visible portion of the sky, allow us to study the influence of the shape of the stone on the formation of the ice foot. Finally, we show that the far-infrared black-body irradiance of the stone itself leads to the formation of a depression surrounding the pedestal.

A wide variety of spectacular structures in which a stone or rock sits on a slender pedestal can be found in nature: hoodoos that consist of a hard stone protecting a narrow column of sedimentary rock from rain-induced erosion (1–3), mushroom rocks whose base is eroded by strong particle-laden winds (4), glacier tables for which a foot of ice resists melting due mostly to thermal insulation provided by a large rock (5–8),^*and Zen stones on frozen lakes in which pebbles rest on delicate ice pedestals, as shown in Fig. 1 A and B. Although these structures differ in the nature of the erosion mechanisms, in timescales (from days for Zen stones to centuries for hoodoos), and in size dimension (from centimeters to tens of meters), the resulting shapes can be strikingly similar. Note also that micro- and nanofabrication processes, largely based upon differential etching of metal or semiconductor substrates, may lead to the formation of micrometer-sized optomechanical resonators with a geometry very similar to that of Zen stones (9, 10).Open in a separate windowFig. 1.Zen stones in nature, in the laboratory, and in numerical simulations. (A) A Zen stone on Lake Baikal (∼16-cm wide) showing a narrow ice pedestal. (B) Zen stones (∼3- to 5-cm wide) in a cave (Lake Baikal) with no direct sunlight. (C) A laboratory-scale experiment using a 30-mm aluminum disk initially resting on a flat ice surface and placed in a lyophilizer for 40 h. (D) A 2D numerical simulation of the phenomenon (the text has details). (A) Image credit: Instagram/zima_landscape. (B) Image credit: A. Yantarev (photographer).Most natural structures in which a stone sits on a pedestal are due to differential melting or mechanical abrasion. However, ice sublimation is known to drive a wide variety of morphologies in astrophysical bodies (11, 12). On Earth, sublimation-driven morphologies are rather scarce, with one well-known example being penitentes (13–15) encountered in the Andes and the Himalayas. However, numerous observations of sublimation-induced patterns have been made throughout the solar system, ranging from penitentes on Pluto (16) to landscape formation on Mars (17–19), Pluto (20), (1) Ceres (21), satellites of Jupiter (22–24), Saturn (25), and comets (26, 27).In this article, we show that the morphogenesis of Zen stones is governed by differential sublimation rates of ice due to the shade provided by the stone, thus reporting a natural occurrence of sublimation-driven pattern formation on Earth. The Zen stones are reproduced in the laboratory (Fig. 1C) using an apparatus that allows for a controlled constant sublimation rate, while numerical simulations based on a geometrical numerical model recover the experimental results and allow us to study the influence of the shape of the stone on the formation of the ice pedestal (Fig. 1D).     

Solute carrier family 3 member 2 (Slc3a2) controls yolk syncytial layer (YSL) formation by regulating microtubule networks in the zebrafish embryo

The yolk syncytial layer (YSL) in the zebrafish embryo is a multinucleated syncytium essential for embryo development, but the molecular mechanisms underlying YSL formation remain largely unknown. Here we show that zebrafish solute carrier family 3 member 2 (Slc3a2) is expressed specifically in the YSL and that slc3a2 knockdown causes severe YSL defects including clustering of the yolk syncytial nuclei and enhanced cell fusion, accompanied by disruption of microtubule networks. Expression of a constitutively active RhoA mimics the YSL phenotypes caused by slc3a2 knockdown, whereas attenuation of RhoA or ROCK activity rescues the slc3a2-knockdown phenotypes. Furthermore, slc3a2 knockdown significantly reduces tyrosine phosphorylation of c-Src, and overexpression of a constitutively active Src restores the slc3a2-knockdown phenotypes. Our data demonstrate a signaling pathway regulating YSL formation in which Slc3a2 inhibits the RhoA/ROCK pathway via phosphorylation of c-Src to modulate YSL microtubule dynamics. This work illuminates processes at a very early stage of zebrafish embryogenesis and more generally informs the mechanism of cell dynamics during syncytium formation.     

Bisphenol A alters the development of the rhesus monkey mammary gland

The xenoestrogen bisphenol A (BPA) used in the manufacturing of various plastics and resins for food packaging and consumer products has been shown to produce numerous endocrine and developmental effects in rodents. Exposure to low doses of BPA during fetal mammary gland development resulted in significant alterations in the gland's morphology that varied from subtle ones observed during the exposure period to precancerous and cancerous lesions manifested in adulthood. This study assessed the effects of BPA on fetal mammary gland development in nonhuman primates. Pregnant rhesus monkeys were fed 400 μg of BPA per kg of body weight daily from gestational day 100 to term, which resulted in 0.68 ± 0.312 ng of unconjugated BPA per mL of maternal serum, a level comparable to that found in humans. At birth, the mammary glands of female offspring were removed for morphological analysis. Morphological parameters similar to those shown to be affected in rodents exposed prenatally to BPA were measured in whole-mounted glands; estrogen receptor (ER) α and β expression were assessed in paraffin sections. Student's t tests for equality of means were used to assess differences between exposed and unexposed groups. The density of mammary buds was significantly increased in BPA-exposed monkeys, and the overall development of their mammary gland was more advanced compared with unexposed monkeys. No significant differences were observed in ER expression. Altogether, gestational exposure to the estrogen-mimic BPA altered the developing mammary glands of female nonhuman primates in a comparable manner to that observed in rodents.     

Hepatic VASP upregulation in rats with secondary biliary cirrhosis by expression in the peribiliary vascular plexus

Introduction/aims: Hepatic disorders in patients and animal models may be characterized by increased ductular proliferation and concomitant expansion of the peribiliary vascular plexus (PBP). VASP plays an essential role for the control of cell assembly during the formation of three-dimensional organ structures in the cardiovascular system. Here, we investigated hepatic VASP expression in response to bile duct ligation (BDL) of rats. Methods: BDL was performed in male rats. Sham-operated rats served as controls. After 4 weeks, hepatic VASP expression was assessed by Western-blot analysis and immunohistochemical staining. Results: Livers of BDL rats showed excessive formation of new bile ducts. VASP mRNA and protein expression was upregulated in whole liver homogenates from BDL rats. This upregulation was located to the expanding PBP in the areas of ductular proliferation. In patients with cirrhosis, hepatic VASP expression was upregulated compared to non-cirrhotic patients. Discussion: VASP may play a role for vascular morphogenesis in the expanding PBP of BDL rats.