Cellular Signaling

Cell communication in the nervous system is finely tuned to respond rapidly to external stimuli, to modify itself according to those stimuli, and to produce more effective responses in the future. The physical basis for this cell communication is the manipulation of ion gradients with ion pumps and channels, chemical neurotransmission, and synaptic plasticity, all of which are discussed in this chapter.

Tubular lysosomes harbor active ion gradients and poise macrophages for phagocytosis

Lysosomes adopt dynamic, tubular states that regulate antigen presentation, phagosome resolution, and autophagy. Tubular lysosomes are studied either by inducing autophagy or by activating immune cells, both of which lead to cell states where lysosomal gene expression differs from the resting state. Therefore, it has been challenging to pinpoint the biochemical properties lysosomes acquire upon tubulation that could drive their functionality. Here we describe a DNA-based assembly that tubulates lysosomes in macrophages without activating them. Proteolytic activity maps at single-lysosome resolution revealed that tubular lysosomes were less degradative and showed proximal to distal luminal pH and Ca2+ gradients. Such gradients had been predicted but never previously observed. We identify a role for tubular lysosomes in promoting phagocytosis and activating MMP9. The ability to tubulate lysosomes without starving or activating immune cells may help reveal new roles for tubular lysosomes.

Ion dynamics at the energy-deprived tripartite synapse

The anatomical and functional organization of neurons and astrocytes at ‘tripartite synapses’ is essential for reliable neurotransmission, which critically depends on ATP. In low energy conditions, synaptic transmission fails, accompanied by a breakdown of ion gradients, changes in membrane potentials and cell swelling. The resulting cellular damage and cell death are causal to the often devastating consequences of an ischemic stroke. The severity of ischemic damage depends on the age and the brain region in which a stroke occurs, but the reasons for this differential vulnerability are far from understood. In the present study, we address this question by developing a comprehensive biophysical model of a glutamatergic synapse to identify key determinants of synaptic failure during energy deprivation. Our model is based on fundamental biophysical principles, includes dynamics of the most relevant ions, i.e., Na+, K+, Ca2+, Cl− and glutamate, and is calibrated with experimental data. It confirms the critical role of the Na+/K+-ATPase in maintaining ion gradients, membrane potentials and cell volumes. Our simulations demonstrate that the system exhibits two stable states, one physiological and one pathological. During energy deprivation, the physiological state may disappear, forcing a transit to the pathological state, which can be reverted when blocking voltage-gated Na+ and K+ channels. Our model predicts that the transition to the pathological state is favoured if the extracellular space fraction is small. A reduction in the extracellular space volume fraction, as, e.g. observed with ageing, will thus promote the brain’s susceptibility to ischemic damage. Our work provides new insights into the brain’s ability to recover from energy deprivation, with translational relevance for diagnosis and treatment of ischemic strokes.

Ion dynamics at the energy-deprived tripartite synapse

The anatomical and functional organization of neurons and astrocytes at ‘tripartite synapses’ is essential for reliable neurotransmission, which critically depends on ATP. In low energy conditions, synaptic transmission fails, accompanied by a breakdown of ion gradients, changes in membrane potentials and cell swelling. The resulting cellular damage and cell death are causal to the often devastating consequences of an ischemic stroke. The severity of ischemic damage depends on the age and the brain region in which a stroke occurs, but the reasons for this differential vulnerability are far from understood. In the present study, we address this question by developing a comprehensive biophysical model of a glutamatergic synapse to identify key determinants of synaptic failure during energy deprivation. Our model is based on fundamental biophysical principles, includes dynamics of the most relevant ions, i.e., Na+, K+, Ca2+, Cl− and glutamate, and is calibrated with experimental data. It confirms the critical role of the Na+/K+-ATPase in maintaining ion gradients, membrane potentials and cell volumes. Our simulations demonstrate that the system exhibits two stable states, one physiological and one pathological. During energy deprivation, the physiological state may disappear, forcing a transit to the pathological state, which can be reverted when blocking voltage-gated Na+ and K+ channels. Our model predicts that the transition to the pathological state is favoured if the extracellular space fraction is small. A reduction in the extracellular space volume fraction, as, e.g. observed with ageing, will thus promote the brain’s susceptibility to ischemic damage. Our work provides new insights into the brain’s ability to recover from energy deprivation, with translational relevance for diagnosis and treatment of ischemic strokes.Author summaryThe brain consumes energy to keep ion concentrations at normal working conditions. In the case of energy deprivation (ED), e.g., during a stroke, synaptic communication fails first. Inspired by our recent experimental work on ED, we formulated a novel computational model to explore initial events during ED. Our model reproduces time courses for several ions from different experimental data. In some cases, the system returns to baseline upon restoring energy supply. In others, we observe that neurons and astrocytes cannot recover accompanied by cell swelling. There is a threshold depending on the depth and duration of ATP depletion differentiating these cases. Also, smaller extracellular spaces hamper recovery more. This result may explain clinical observations of increased vulnerability to stroke as the size of the extracellular space shrinks with ageing.

Paracellular Filtration Secretion Driven by Mechanical Force Contributes to Small Intestinal Fluid Dynamics

Studies of fluid secretion by the small intestine are dominated by the coupling with ATP-dependent generation of ion gradients, whereas the contribution of filtration secretion has been overlooked, possibly by the lack of a known mechanistic basis. We measured apical fluid flow and generation of hydrostatic pressure gradients by epithelia of cultured mouse enterocytes, Caco-2 and T-84 cells, and fibroblasts exposed to mechanical force provided by vigorous aeration and in response to ion gradients, inhibitors of ion channels and transporters and in vitro using intact mouse and rat small intestine. We describe herein a paracellular pathway for unidirectional filtration secretion that is driven by mechanical force, requires tight junctions, is independent of ionic and osmotic gradients, generates persistent hydrostatic pressure gradients, and would contribute to the fluid shifts that occur during digestion and diarrhea. Zinc inhibits the flow of fluid and the paracellular marker fluorescein isothyocyanate conjugated dextran (MW = 4 kD) across epithelia of cultured enterocytes (>95%; p < 0.001) and intact small intestine (>40%; p = 0.03). We propose that mechanical force drives fluid secretion through the tight junction complex via a “one-way check valve” that can be regulated. This pathway of filtration secretion complements chloride-coupled fluid secretion during high-volume fluid flow. The role of filtration secretion in the genesis of diarrhea in intact animals needs further study. Our findings may explain a potential linkage between intestinal motility and intestinal fluid dynamics.

Tubular lysosomes harbor active ion gradients and poise macrophages for phagocytosis

Lysosomes adopt dynamic, tubular states that regulate antigen presentation, phagosome resolution and autophagy. To date, tubular lysosomes have been studied either by inducing autophagy or by activating immune cells, both of which lead to cell states where lysosomal gene expression differs from the resting state. Therefore, it has been challenging to pinpoint the specific biochemical properties lysosomes acquire upon tubulation that could drive their functionality. We describe a DNA-based assembly that tubulates lysosomes in macrophages without activating them. Lumenal proteolytic activity maps at single lysosome resolution revealed that tubular lysosomes were less degradative. Further, they showed striking proximal to distal lumenal pH and Ca2+ gradients. Such gradients had been predicted, but never previously observed. We now identify a role for tubular lysosomes whereby they poise resting macrophages for phagocytosis. The ability to tubulate lysosomes without having to starve or activate immune cells may help reveal new roles for tubular lysosomes.

Semipermeable Mixed Phospholipid-Fatty Acid Membranes Exhibit K+/Na+ Selectivity in the Absence of Proteins

Two important ions, K+ and Na+, are unequally distributed across the contemporary phospholipid-based cell membrane because modern cells evolved a series of sophisticated protein channels and pumps to maintain ion gradients. The earliest life-like entities or protocells did not possess either ion-tight membranes or ion pumps, which would result in the equilibration of the intra-protocellular K+/Na+ ratio with that in the external environment. Here, we show that the most primitive protocell membranes composed of fatty acids, that were initially leaky, would eventually become less ion permeable as their membranes evolved towards having increasing phospholipid contents. Furthermore, these mixed fatty acid-phospholipid membranes selectively retain K+ but allow the passage of Na+ out of the cell. The K+/Na+ selectivity of these mixed fatty acid-phospholipid semipermeable membranes suggests that protocells at intermediate stages of evolution could have acquired electrochemical K+/Na+ ion gradients in the absence of any macromolecular transport machinery or pumps, thus potentially facilitating rudimentary protometabolism.

Neural effects of elevated CO2 in fish may be amplified by a vicious cycle

Lay summaries: The altered behaviours of fish exposed to elevated CO2 have been linked to changes in ion gradients and neurotransmitter function. To explain how relatively small changes in ion concentrations could have such profound neural effects, we propose that a vicious cycle can be triggered that amplifies the initial disturbance.