Division site selection linked to inherited cell surface wave troughs in mycobacteria

Cell division is tightly controlled in space and time to maintain cell size and ploidy within narrow bounds. In bacteria, the canonical Minicell (Min) and nucleoid occlusion (Noc) systems together ensure that division is restricted to midcell after completion of chromosome segregation1. It is unknown how division site selection is controlled in bacteria that lack homologues of the Min and Noc proteins, including mycobacteria responsible for tuberculosis and other chronic infections2. Here, we use correlated optical and atomic-force microscopy3,4 to demonstrate that morphological landmarks (waveform troughs) on the undulating surface of mycobacterial cells correspond to future sites of cell division. Newborn cells inherit wave troughs from the (grand)mother cell and ultimately divide at the centre-most wave trough, making these morphological features the earliest known landmark of future division sites. In cells lacking the chromosome partitioning (Par) system, missegregation of chromosomes is accompanied by asymmetric cell division at off-centre wave troughs, resulting in the formation of anucleate cells. These results demonstrate that inherited morphological landmarks and chromosome positioning together restrict mycobacterial division to the midcell position

Hybrid hydrodynamic characteristic for hydrocephalus valve: A numerical investigation using electrical equivalent networks

International audienceNumerical simulations based on the classical Marmarou’s model have been carried out to analyse the dynamics of hydrocephalus valves. The evolution of the intracranial pressure in various case studies has been determined with a specific focus on flow control valves. It has been shown that their capability to prevent postural under- and over-drainage may be significantly altered by non-idealities of their hydrodynamic characteristics and by the inter-individual variability of the cerebrospinal fluid production rate. The use of microtechnology to improve the flow rate accuracy also enables the possibility to get original designs that are desirable to address specific restrictions of use associated with flow-control valves, in particular for patients exhibiting very high resistance to cerebrospinal fluid reabsorption. A new hybrid hydrodynamic characteristic of a hydrocephalus valve is proposed to stabilize the intracranial pressure. This new passive valve is equivalent to two pressure regulators at high and low relative pressures, corresponding respectively to upright and decubitus positions. The device is able to automatically switch from one configuration to the other as a function of the postural change. Numerical simulations suggest that this new hybrid valve should combine the advantages of both differential and flow control valves

Design of a Passive Flow Regulator Using a Genetic Algorithm

Passive flow regulators are usually intended to deliver or drain a fluid at a constant rate independently from pressure variations. Microfluidic devices made of a stack of two plates are considered here: the first plate comprises a flexible silicon membrane having through holes while the second plate is a rigid substrate with a cavity, an outlet hole and pillars aligned with the through holes of the membrane. The liquid flows through the holes etched in the membrane and through the small gap between the bottom of the membrane and the pillars: each hole can therefore be considered as a valve which progressively closes as the pressure increases, thus leading to a non-linear fluidic behaviour. FEM simulations have been performed to ensure a constant flow rate in the specified range of pressure. To make the design reliable, the device characteristics have been optimized using an evolutionary algorithm. The fitness function notably takes into account machining and alignment tolerances. Typical designs dedicated to drug delivery and hydrocephalus treatment are discussed

Volcano-Shaped Scanning Probe Microscopy Probe for Combined Force-Electrogram Recordings from Excitable Cells

Atomic force microscopy based approaches have led to remarkable advances in the field of mechanobiology. However, linking the mechanical cues to biological responses requires complementary techniques capable of recording these physiological characteristics. In this study, we present an instrument for combined optical, force, and electrical measurements based on a novel type of scanning probe microscopy cantilever composed of a protruding volcano-shaped nanopattemed microelectrode (nanovolcano probe) at the tip of a suspended microcantilever. This probe enables simultaneous force and electrical recordings from single cells. Successful impedance measurements on mechanically stimulated neonatal rat cardiomyocytes in situ were achieved using these nanovolcano probes. Furthermore, proof of concept experiments demonstrated that extracellular field potentials (electrogram) together with contraction displacement curves could simultaneously be recorded. These features render the nanovolcano probe especially suited for mechanobiological studies aiming at linking mechanical stimuli to electrophysiological responses of single cells

Passive flow control valve for protein delivery

Passive ow control valves are usually intended to deliver or drain a uid at a constant rate independently of pressure variations. Micro uidic devices made of a stack of two plates are considered here: the rst plate comprises a exible silicon membrane having through holes while the second plate is a rigid substrate with a cavity, an outlet hole and pillars aligned with the through holes of the membrane. The liquid ows through the holes etched in the membrane and through the gap be- tween the membrane and the pillars. Each gap can be considered as a valve which progressively closes as the pressure increases. Numerical modelling of the uid dynamics inside the device associated with FEM simulations of the membrane dis- tortion have been performed to design a device that exhibits a constant ow rate in a speci ed range of pressure. To make the design more reliable, the device charac- teristics have been optimized using a genetic algorithm, the tness function taking notably into account machining and alignment tolerances. This algorithm has been nally used to design ow control valves for wearable injectors dedicated to the in- fusion of viscous drug formulations (hyaluronic acid, adalimumab, golimumab ...) at high pressure. Prototypes have been characterized using solutions of 12 and 24 cP. It has been demonstrated experimentally that this technology is suitable to passively infuse biological products at ow rates up to 1 mL/min. The numerical model has then been re ned further so as to obtain a good correlation with experimental data

Overlapping and essential roles for molecular and mechanical mechanisms in mycobacterial cell division

Mechanisms to control cell division are essential for cell proliferation and survival. Bacterial cell growth and division require the coordinated activity of peptidoglycan synthases and hydrolytic enzymes to maintain mechanical integrity of the cell wall. Recent studies suggest that cell separation is governed by mechanical forces. How mechanical forces interact with molecular mechanisms to control bacterial cell division in space and time is poorly understood. Here we use a combination of atomic force microscope imaging, nanomechanical mapping and nanomanipulation to show that enzymatic activity and mechanical forces serve overlapping and essential roles in mycobacterial cell division. We find that mechanical stress gradually accumulates in the cell wall, concentrated at the future division site, culminating in rapid (millisecond) cleavage of nascent sibling cells. Inhibiting cell wall hydrolysis delays cleavage; conversely, locally increasing cell wall stress causes instantaneous and premature cleavage. Cells deficient in peptidoglycan hydrolytic activity fail to locally decrease their cell wall strength and undergo natural cleavage, instead forming chains of non-growing cells. Cleavage of these cells can be mechanically induced by local application of stress with an atomic force microscope. These findings establish a direct link between actively controlled molecular mechanisms and passively controlled mechanical forces in bacterial cell division

A biphasic growth model for cell pole elongation in mycobacteria

Mycobacteria grow by inserting new cell wall material in discrete zones at the cell poles. This pattern implies that polar growth zones must be assembled de novo at each division, but the mechanisms that control the initiation of new pole growth are unknown. Here, we combine time-lapse optical and atomic force microscopy to measure single-cell pole growth in mycobacteria with nanometer-scale precision. We show that single-cell growth is biphasic due to a lag phase of variable duration before the new pole transitions from slow to fast growth. This transition and cell division are independent events. The difference between the lag and interdivision times determines the degree of single-cell growth asymmetry, which is high in fast-growing species and low in slow-growing species. We propose a biphasic growth model that is distinct from previous unipolar and bipolar models and resembles “new end take off” (NETO) dynamics of polar growth in fission yeast

Mechanopathology of biofilm-like Mycobacterium tuberculosis cords.

Mycobacterium tuberculosis (Mtb) cultured axenically without detergent forms biofilm-like cords, a clinical identifier of virulence. In lung-on-chip (LoC) and mouse models, cords in alveolar cells contribute to suppression of innate immune signaling via nuclear compression. Thereafter, extracellular cords cause contact-dependent phagocyte death but grow intercellularly between epithelial cells. The absence of these mechanopathological mechanisms explains the greater proportion of alveolar lesions with increased immune infiltration and dissemination defects in cording-deficient Mtb infections. Compression of Mtb lipid monolayers induces a phase transition that enables mechanical energy storage. Agent-based simulations demonstrate that the increased energy storage capacity is sufficient for the formation of cords that maintain structural integrity despite mechanical perturbation. Bacteria in cords remain translationally active despite antibiotic exposure and regrow rapidly upon cessation of treatment. This study provides a conceptual framework for the biophysics and function in tuberculosis infection and therapy of cord architectures independent of mechanisms ascribed to single bacteria