Kate Poole

The Molecular and Cellular Identity of Peripheral Osmoreceptors

In mammals, the osmolality of the extracellular fluid (ECF) is highly stable despite radical changes in salt/water intake and excretion. Afferent systems are required to detect hypo- or hyperosmotic shifts in the ECF to trigger homeostatic control of osmolality. In humans, a pressor reflex is triggered by simply drinking water which may be mediated by peripheral osmoreceptors. Here, we identified afferent neurons in the thoracic dorsal root ganglia (DRG) of mice that innervate hepatic blood vessels and detect physiological hypo-osmotic shifts in blood osmolality. Hepatic sensory neurons are equipped with an inward current that faithfully transduces graded changes in osmolality within the physiological range (~15 mOsm). In mice lacking the osmotically activated ion channel, TRPV4, hepatic sensory neurons no longer exhibit osmosensitive inward currents and activation of peripheral osmoreceptors in vivo is abolished. We have thus identified a new population of sensory neurons that transduce ongoing changes in hepatic osmolality.

Probing cellular microenvironments and tissue remodeling by atomic force microscopy.

The function of cells is strongly determined by the properties of their extracellular microenvironment. Biophysical parameters like environmental stiffness and fiber orientation in the surrounding matrix are important determinants of cell adhesion and migration. Processes like tissue maintenance, wound repair, cancer cell invasion, and morphogenesis depend critically on the ability of cells to actively sense and remodel their surroundings. Pericellular proteolytic activity and adaptation of migration tactics to the environment are strategies to achieve this aim. Little is known about the distinct regulatory mechanisms that are involved in these processes. The system’s critical biophysical and biochemical determinants are well accessible by atomic force microscopy (AFM), a unique tool for functional, nanoscale probing and morphometric, high-resolution imaging of processes in live cells. This review highlights common principles of tissue remodeling and focuses on application examples of different AFM techniques, for example elasticity mapping, the combination of AFM and fluorescence microscopy, the morphometric imaging of proteolytic activity, and force spectroscopy applications of single molecules or individual cells. To achieve a more complete understanding of the processes underlying the interaction of cells with their environments, the combination of AFM force spectroscopy experiments will be essential.

Tuning Piezo ion channels to detect molecular-scale movements relevant for fine touch

In sensory neurons, mechanotransduction is sensitive, fast and requires mechanosensitive ion channels. Here we develop a new method to directly monitor mechanotransduction at defined regions of the cell-substrate interface. We show that molecular-scale (~13 nm) displacements are sufficient to gate mechanosensitive currents in mouse touch receptors. Using neurons from knockout mice, we show that displacement thresholds increase by one order of magnitude in the absence of stomatin-like protein 3 (STOML3). Piezo1 is the founding member of a class of mammalian stretch-activated ion channels, and we show that STOML3, but not other stomatin-domain proteins, brings the activation threshold for Piezo1 and Piezo2 currents down to ~10 nm. Structure–function experiments localize the Piezo modulatory activity of STOML3 to the stomatin domain, and higher-order scaffolds are a prerequisite for function. STOML3 is the first potent modulator of Piezo channels that tunes the sensitivity of mechanically gated channels to detect molecular-scale stimuli relevant for fine touch.

The effect of raft lipid depletion on microvilli formation in MDCK cells, visualized by atomic force microscopy.

We have investigated whether raft lipids of Madin–Darby canine kidney (MDCK) cells play any role in microvilli maintenance using a combination of atomic force microscopy (AFM) and laser scanning confocal microscopy. MDCK cells were treated to reduce the amount of sphingolipids, cholesterol, or both and subsequently imaged, in buffer solution, using AFM. It was observed that inhibition of either sphingolipid or cholesterol biosynthesis led to a reduction in the number of microvilli on the surface of MDCK cells. However, this effect was not uniform across the monolayer, with some cells resembling those in untreated controls. The subsequent extraction of cholesterol from cells grown in the presence of inhibitors led to a further reduction in microvilli on the surface of the cells and, in some cases, resulted in monolayers devoid of full length microvilli. Significantly, smaller spikes were observed on the surface of the smoother cells.

Stomatin-domain proteins

The stomatin-domain defines a family of proteins that are found in all classes of life. The ubiquity of stomatin-family proteins and their high degree of homology suggest that they have a unifying cellular function, which has yet to be defined. The five stomatin family proteins in mammals show varying expression patterns and different sub-cellular distributions. In surveying the relevant literature, three common themes emerge; stomatin family members are oligomeric; they mostly localise to membrane domains; and in many cases, they have been shown to modulate ion channel activity. How oligomerisation and membrane localisation contribute to the modulation of channel function is unclear to date. Future studies into the precise structure and mechanism of stomatin-like proteins need to address these important questions to clarify the detailed cellular function of stomatin-domain containing proteins.

Laminin-332 coordinates mechanotransduction and growth cone bifurcation in sensory neurons.

Laminin-332 is a major component of the dermo-epidermal skin basement membrane and maintains skin integrity. The transduction of mechanical force into electrical signals by sensory endings in the skin requires mechanosensitive channels. We found that mouse epidermal keratinocytes produce a matrix that is inhibitory for sensory mechanotransduction and that the active molecular component is laminin-332. Substrate-bound laminin-332 specifically suppressed one type of mechanosensitive current (rapidly adapting) independently of integrin-receptor activation. This mechanotransduction suppression could be exerted locally and was mediated by preventing the formation of protein tethers necessary for current activation. We also found that laminin-332 could locally control sensory axon branching behavior. Loss of laminin-332 in humans led to increased sensory terminal branching and may lead to a de-repression of mechanosensitive currents. These previously unknown functions for this matrix molecule may explain some of the extreme pain experienced by individuals with epidermolysis bullosa who are deficient in laminin-332.

A stomatin dimer modulates the activity of acid‐sensing ion channels

Stomatin proteins oligomerize at membranes and have been implicated in ion channel regulation and membrane trafficking. To obtain mechanistic insights into their function, we determined three crystal structures of the conserved stomatin domain of mouse stomatin that assembles into a banana‐shaped dimer. We show that dimerization is crucial for the repression of acid‐sensing ion channel 3 (ASIC3) activity. A hydrophobic pocket at the inside of the concave surface is open in the presence of an internal peptide ligand and closes in the absence of this ligand, and we demonstrate a function of this pocket in the inhibition of ASIC3 activity. In one crystal form, stomatin assembles via two conserved surfaces into a cylindrical oligomer, and these oligomerization surfaces are also essential for the inhibition of ASIC3‐mediated currents. The assembly mode of stomatin uncovered in this study might serve as a model to understand oligomerization processes of related membrane‐remodelling proteins, such as flotillin and prohibitin.

Flexible, actin-based ridges colocalise with the β1 integrin on the surface of melanoma cells

Using a combination of laser-scanning confocal microscopy and atomic force microscopy, we have identified flexible, actin-based structures on the surface of cells derived from the vertical growth phase of melanoma progression. These flexible structures, lacking on the surface of mature melanocytes, were observed on the surface of all four melanoma cell lines tested. Further investigation revealed that the β1 integrin colocalises with these actin-based ridges on the cell surface, whereas β1 integrin distribution in melanocytes did not correlate with actin-based structures. Fibronectin staining on the surface of melanoma cells was partially codistributed with the ridges. The combination of structural information derived from atomic force microscopy images and fluorescent imaging of the distribution of labelled proteins involved in invasion and metastasis has allowed us to identify a common feature that may be involved in disease progression, at the surface of vertical growth phase melanoma cells, despite the known variation in genetic composition of melanoma.

Direct measurement of TRPV4 and PIEZO1 activity reveals multiple mechanotransduction pathways in chondrocytes

The joints of mammals are lined with cartilage, comprised of individual chondrocytes embedded in a specialized extracellular matrix. Chondrocytes experience a complex mechanical environment and respond to changing mechanical loads in order to maintain cartilage homeostasis. It has been proposed that mechanically gated ion channels are of functional importance in chondrocyte mechanotransduction; however, direct evidence of mechanical current activation in these cells has been lacking. We have used high-speed pressure clamp and elastomeric pillar arrays to apply distinct mechanical stimuli to primary murine chondrocytes, stretch of the membrane and deflection of cell-substrate contacts points, respectively. Both TRPV4 and PIEZO1 channels contribute to currents activated by stimuli applied at cell-substrate contacts but only PIEZO1 mediates stretch-activated currents. These data demonstrate that there are separate, but overlapping, mechanoelectrical transduction pathways in chondrocytes. DOI: http://dx.doi.org/10.7554/eLife.21074.001

Regulation of ASIC channels by a stomatin/STOML3 complex located in a mobile vesicle pool in sensory neurons

A complex of stomatin-family proteins and acid-sensing (proton-gated) ion channel (ASIC) family members participate in sensory transduction in invertebrates and vertebrates. Here, we have examined the role of the stomatin-family protein stomatin-like protein-3 (STOML3) in this process. We demonstrate that STOML3 interacts with stomatin and ASIC subunits and that this occurs in a highly mobile vesicle pool in dorsal root ganglia (DRG) neurons and Chinese hamster ovary cells. We identify a hydrophobic region in the N-terminus of STOML3 that is required for vesicular localization of STOML3 and regulates physical and functional interaction with ASICs. We further characterize STOML3-containing vesicles in DRG neurons and show that they are Rab11-positive, but not part of the early-endosomal, lysosomal or Rab14-dependent biosynthetic compartment. Moreover, uncoupling of vesicles from microtubules leads to incorporation of STOML3 into the plasma membrane and increased acid-gated currents. Thus, STOML3 defines a vesicle pool in which it associates with molecules that have critical roles in sensory transduction. We suggest that the molecular features of this vesicular pool may be characteristic of a ‘transducosome’ in sensory neurons.