Atomic force microscopy-based mechanobiology

Mechanobiology emerges at the crossroads of medicine, biology, biophysics and engineering and describes how the responses of proteins, cells, tissues and organs to mechanical cues contribute to development, differentiation, physiology and disease. The grand challenge in mechanobiology is to quantify how biological systems sense, transduce, respond and apply mechanical signals. Over the past three decades, atomic force microscopy (AFM) has emerged as a key platform enabling the simultaneous morphological and mechanical characterization of living biological systems. In this Review, we survey the basic principles, advantages and limitations of the most common AFM modalities used to map the dynamic mechanical properties of complex biological samples to their morphology. We discuss how mechanical properties can be directly linked to function, which has remained a poorly addressed issue. We outline the potential of combining AFM with complementary techniques, including optical microscopy and spectroscopy of mechanosensitive fluorescent constructs, super-resolution microscopy, the patch clamp technique and the use of microstructured and fluidic devices to characterize the 3D distribution of mechanical responses within biological systems and to track their morphology and functional state.Peer ReviewedPostprint (published version

GenEPi: Piezo1-based fluorescent reporter for visualizing mechanical stimuli with high spatiotemporal resolution

Mechanosensing is a ubiquitous process to translate external mechanical stimuli into biological responses during development, homeostasis, and disease. However, non-invasive investigation of cellular mechanosensing in complex and intact live tissue remains challenging. Here, we developed GenEPi, a genetically-encoded fluorescent intensiometric reporter for mechanical stimuli based on Piezo1, an essential mechanosensitive ion channel found in vertebrates. We show that GenEPi has high specificity and spatiotemporal resolution for Piezo1-dependent mechanical stimuli, exemplified by resolving repetitive mechanical stimuli of spontaneously contracting cardiomyocytes within microtissues, in a non-invasive manner

Restoration of Visual Function by Expression of a Light-Gated Mammalian Ion Channel in Retinal Ganglion Cells or ON-Bipolar Cells

Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second- and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration (rd1) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0460). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform light-evoked firing when LiGluR-MAG0460 was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0460 was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0460 in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rod-cone dystrophy dog model of blindness, LiGluR-MAG0460 in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0460 was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation

Restoration of patterned vision with an engineered photoactivatable G protein-coupled receptor

Retinitis pigmentosa results in blindness due to degeneration of photoreceptors, but spares other retinal cells, leading to the hope that expression of light-activated signaling proteins in the surviving cells could restore vision. We used a retinal G protein-coupled receptor, mGluR2, which we chemically engineered to respond to light. In retinal ganglion cells (RGCs) of blind rd1 mice, photoswitch-charged mGluR2 ("SNAG-mGluR2") evoked robust OFF responses to light, but not in wild-type retinas, revealing selectivity for RGCs that have lost photoreceptor input. SNAG-mGluR2 enabled animals to discriminate parallel from perpendicular lines and parallel lines at varying spacing. Simultaneous viral delivery of the inhibitory SNAG-mGluR2 and excitatory light-activated ionotropic glutamate receptor LiGluR yielded a distribution of expression ratios, restoration of ON, OFF and ON-OFF light responses and improved visual acuity. Thus, SNAG-mGluR2 restores patterned vision and combinatorial light response diversity provides a new logic for enhanced-acuity retinal prosthetics

Optical Control of Metabotropic Glutamate Receptors

G-protein coupled receptors (GPCRs), the largest family of membrane signaling proteins, respond to neurotransmitters, hormones and small environmental molecules. The neuronal function of many GPCRs has been difficult to resolve because of an inability to gate them with subtype-specificity, spatial precision, speed and reversibility. To address this, we developed an approach for opto-chemical engineering native GPCRs. We applied this to the metabotropic glutamate receptors (mGluRs) to generate light-agonized and light-antagonized “LimGluRs”. The light-agonized “LimGluR2”, on which we focused, is fast, bistable, and supports multiple rounds of on/off switching. Light gates two of the primary neuronal functions of mGluR2: suppression of excitability and inhibition of neurotransmitter release. The light-antagonized “LimGluR2block” can be used to manipulate negative feedback of synaptically released glutamate on transmitter release. We generalize the optical control to two additional family members: mGluR3 and 6. The system works in rodent brain slice and in zebrafish in vivo, where we find that mGluR2 modulates the threshold for escape behavior. These light-gated mGluRs pave the way for determining the roles of mGluRs in synaptic plasticity, memory and disease

Vision restoration in animal models of human blindness using natural and engineered light-gated receptors

Most inherited forms of human blindness are caused by mutations that lead to photoreceptor cell death, but spare the inner retina, providing an opportunity for treatment. With the help of azobenzene-derived chemicals that we call ‘photoswitches’, I engineered light gated receptors to be applied as therapeutics towards vision restoration in animal models of human blindness. The first part of my thesis describes this work.Next, I targeted expression of natural and engineered light-gated proteins to the remaining neurons of the retina, using viruses as gene delivery vehicles. I asked if these cells would then function as the new photoreceptors and if they would be able to drive visual responses. The quick answer is: yes, they do. In blind mice, I compared the ability of different target cells to act as new photoreceptors. Installing light sensors downstream in retinal ganglion cells lead to robust and uniform responses, whereas expression upstream in bipolar cells lead to more diverse activity patterns in response to light. I characterized mammalian proteins as optical actuators and found that light gated ion channels drive fast responses but require very high light intensities whereas G-protein coupled receptors are about 1000x more sensitive to light but at the cost of slow kinetics. I then further extended our studies to dogs and were able to show that our treatment restored light responses in blind rcd1 dog retinas in vitro and was safe and well tolerated in vivo. My results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation. These findings are summarized in the second part of my thesis.Finally, I explored non-invasive approaches to restore a sense of ‘space’ and enable navigation for the blind. Towards this end, I helped design and prototype a sensory substitution device, the ‘sonic eye’. This device is inspired by bats and human echolocators and it allows users to ‘see with sound’. This work is described in the last part of my thesis

Vision restoration in animal models of human blindness using natural and engineered light-gated receptors

Most inherited forms of human blindness are caused by mutations that lead to photoreceptor cell death, but spare the inner retina, providing an opportunity for treatment. With the help of azobenzene-derived chemicals that we call ‘photoswitches’, I engineered light gated receptors to be applied as therapeutics towards vision restoration in animal models of human blindness. The first part of my thesis describes this work.Next, I targeted expression of natural and engineered light-gated proteins to the remaining neurons of the retina, using viruses as gene delivery vehicles. I asked if these cells would then function as the new photoreceptors and if they would be able to drive visual responses. The quick answer is: yes, they do. In blind mice, I compared the ability of different target cells to act as new photoreceptors. Installing light sensors downstream in retinal ganglion cells lead to robust and uniform responses, whereas expression upstream in bipolar cells lead to more diverse activity patterns in response to light. I characterized mammalian proteins as optical actuators and found that light gated ion channels drive fast responses but require very high light intensities whereas G-protein coupled receptors are about 1000x more sensitive to light but at the cost of slow kinetics. I then further extended our studies to dogs and were able to show that our treatment restored light responses in blind rcd1 dog retinas in vitro and was safe and well tolerated in vivo. My results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation. These findings are summarized in the second part of my thesis.Finally, I explored non-invasive approaches to restore a sense of ‘space’ and enable navigation for the blind. Towards this end, I helped design and prototype a sensory substitution device, the ‘sonic eye’. This device is inspired by bats and human echolocators and it allows users to ‘see with sound’. This work is described in the last part of my thesis

Vision restoration in animal models of human blindness using natural and engineered light-gated receptors

Most inherited forms of human blindness are caused by mutations that lead to photoreceptor cell death, but spare the inner retina, providing an opportunity for treatment. With the help of azobenzene-derived chemicals that we call ‘photoswitches’, I engineered light gated receptors to be applied as therapeutics towards vision restoration in animal models of human blindness. The first part of my thesis describes this work.Next, I targeted expression of natural and engineered light-gated proteins to the remaining neurons of the retina, using viruses as gene delivery vehicles. I asked if these cells would then function as the new photoreceptors and if they would be able to drive visual responses. The quick answer is: yes, they do. In blind mice, I compared the ability of different target cells to act as new photoreceptors. Installing light sensors downstream in retinal ganglion cells lead to robust and uniform responses, whereas expression upstream in bipolar cells lead to more diverse activity patterns in response to light. I characterized mammalian proteins as optical actuators and found that light gated ion channels drive fast responses but require very high light intensities whereas G-protein coupled receptors are about 1000x more sensitive to light but at the cost of slow kinetics. I then further extended our studies to dogs and were able to show that our treatment restored light responses in blind rcd1 dog retinas in vitro and was safe and well tolerated in vivo. My results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation. These findings are summarized in the second part of my thesis.Finally, I explored non-invasive approaches to restore a sense of ‘space’ and enable navigation for the blind. Towards this end, I helped design and prototype a sensory substitution device, the ‘sonic eye’. This device is inspired by bats and human echolocators and it allows users to ‘see with sound’. This work is described in the last part of my thesis