Showing 1 - 11 of 11 results
Reversible photoregulation of cell-cell adhesions with opto-E-cadherin.
E-cadherin-based cell-cell adhesions are dynamically and locally regulated in many essential processes, including embryogenesis, wound healing and tissue organization, with dysregulation manifesting as tumorigenesis and metastasis. However, the lack of tools that would provide control of the high spatiotemporal precision observed with E-cadherin adhesions hampers investigation of the underlying mechanisms. Here, we present an optogenetic tool, opto-E-cadherin, that allows reversible control of E-cadherin-mediated cell-cell adhesions with blue light. With opto-E-cadherin, functionally essential calcium binding is photoregulated such that cells expressing opto-E-cadherin at their surface adhere to each other in the dark but not upon illumination. Consequently, opto-E-cadherin provides remote control over multicellular aggregation, E-cadherin-associated intracellular signalling and F-actin organization in 2D and 3D cell cultures. Opto-E-cadherin also allows switching of multicellular behaviour between single and collective cell migration, as well as of cell invasiveness in vitro and in vivo. Overall, opto-E-cadherin is a powerful optogenetic tool capable of controlling cell-cell adhesions at the molecular, cellular and behavioural level that opens up perspectives for the study of dynamics and spatiotemporal control of E-cadherin in biological processes.
Spatiotemporally confined red light-controlled gene delivery at single-cell resolution using adeno-associated viral vectors.
Methodologies for the controlled delivery of genetic information into target cells are of utmost importance for genetic engineering in both fundamental and applied research. However, available methods for efficient gene transfer into user-selected or even single cells suffer from low throughput, the need for complicated equipment, high invasiveness, or side effects by off-target viral uptake. Here, we engineer an adeno-associated viral (AAV) vector system that transfers genetic information into native target cells upon illumination with cell-compatible red light. This OptoAAV system allows adjustable and spatially resolved gene transfer down to single-cell resolution and is compatible with different cell lines and primary cells. Moreover, the sequential application of multiple OptoAAVs enables spatially resolved transduction with different transgenes. The approach presented is likely extendable to other classes of viral vectors and is expected to foster advances in basic and applied genetic research.
Spatiotemporal Control Over Multicellular Migration Using Green Light Reversible Cell–Cell Interactions.
The regulation of cell–cell adhesions in space and time plays a crucial role in cell biology, especially in the coordination of multicellular behavior. Therefore, tools that allow for the modulation of cell–cell interactions with high precision are of great interest to a better understanding of their roles and building tissue‐like structures. Herein, the green light‐responsive protein CarH is expressed at the plasma membrane of cells as an artificial cell adhesion receptor, so that upon addition of its cofactor vitamin B12 specific cell–cell interactions form and lead to cell clustering in a concentration‐dependent manner. Upon green light illumination, the CarH based cell–cell interactions disassemble and allow for their reversion with high spatiotemporal control. Moreover, these artificial cell–cell interactions impact cell migration, as observed in a wound‐healing assay. When the cells interact with each other in the presence of vitamin B12 in the dark, the cells form on a solid front and migrate collectively; however, under green light illumination, individual cells migrate randomly out of the monolayer. Overall, the possibility of precisely controlling cell–cell interactions and regulating multicellular behavior is a potential pathway to gaining more insight into cell–cell interactions in biological processes.
Orthogonal Blue and Red Light Controlled Cell-Cell Adhesions Enable Sorting-out in Multicellular Structures.
The self-assembly of different cell types into multicellular structures and their organization into spatiotemporally controlled patterns are both challenging and extremely powerful to understand how cells function within tissues and for bottom-up tissue engineering. Here, we not only independently control the self-assembly of two cell types into multicellular architectures with blue and red light, but also achieve their self-sorting into distinct assemblies. This required developing two cell types that form selective and homophilic cell-cell interactions either under blue or red light using photoswitchable proteins as artificial adhesion molecules. The interactions were individually triggerable with different colors of light, reversible in the dark, and provide noninvasive and temporal control over the cell-cell adhesions. In mixtures of the two cells, each cell type self-assembled independently upon orthogonal photoactivation, and cells sorted out into separate assemblies based on specific self-recognition. These self-sorted multicellular architectures provide us with a powerful tool for producing tissue-like structures from multiple cell types and investigate principles that govern them.
The importance of cell-cell interaction dynamics in bottom-up tissue engineering: Concepts of colloidal self-assembly in the fabrication of multicellular architectures.
Building tissue from cells as the basic building block based on principles of self-assembly is a challenging and promising approach. Understanding how far principles of self-assembly and self-sorting known for colloidal particles apply to cells remains unanswered. In this study, we demonstrate that not just controlling the cell-cell interactions but also their dynamics is a crucial factor that determines the formed multicellular structure, using photoswitchable interactions between cells that are activated with blue light and reverse in the dark. Tuning dynamics of the cell-cell interactions by pulsed light activation, results in multicellular architectures with different sizes and shapes. When the interactions between cells are dynamic compact and round multicellular clusters under thermodynamic control form, while otherwise branched and lose aggregates under kinetic control assemble. These structures parallel what is known for colloidal assemblies under reaction and diffusion limited cluster aggregation, respectively. Similarly, dynamic interactions between cells are essential for cells to self-sort into distinct groups. Using four different cell types, which expressed two orthogonal cell-cell interaction pairs, the cells sorted into two separate assemblies. Bringing concepts of colloidal self-assembly to bottom-up tissue engineering provides a new theoretical framework and will help in the design of more predictable tissue-like structures.
Red/Far-Red Light Switchable Cargo Attachment and Release in Bacteria-Driven Microswimmers.
In bacteria-driven microswimmers, i.e., bacteriabots, artificial cargos are attached to flagellated chemotactic bacteria for active delivery with potential applications in biomedical technology. Controlling when and where bacteria bind and release their cargo is a critical step for bacteriabot fabrication and efficient cargo delivery/deposition at the target site. Toward this goal, photoregulating the cargo integration and release in bacteriabots using red and far-red light, which are noninvasive stimuli with good tissue penetration and provide high spatiotemporal control, is proposed. In the bacteriabot design, the surfaces of E. coli and microsized model cargo particles with the proteins PhyB and PIF6, which bind to each other under red light and dissociate from each other under far-red light are functionalized. Consequently, the engineered bacteria adhere and transport the model cargo under red light and release it on-demand upon far-red light illumination due to the photoswitchable PhyB-PIF6 protein interaction. Overall, the proof-of-concept for red/far-red light switchable bacteriabots, which opens new possibilities in the photoregulation in biohybrid systems for bioengineering, targeted drug delivery, and lab-on-a-chip devices, is demonstrated.
Blue Light Switchable Cell–Cell Interactions Provide
Reversible and Spatiotemporal Control Towards
Bottom-Up Tissue Engineering.
Controlling cell–cell interactions is central for understanding key cellular
processes and bottom-up tissue assembly from single cells. The challenge is
to control cell–cell interactions dynamically and reversibly with high spati-
otemporal precision noninvasively and sustainably. In this study, cell–cell
interactions are controlled with visible light using an optogenetic approach by
expressing the blue light switchable proteins CRY2 or CIBN on the surfaces of
cells. CRY2 and CIBN expressing cells form specific heterophilic interactions
under blue light providing precise control in space and time. Further, these
interactions are reversible in the dark and can be repeatedly and dynamically
switched on and off. Unlike previous approaches, these genetically encoded
proteins allow for long-term expression of the interaction domains and
respond to nontoxic low intensity blue light. In addition, these interactions
are suitable to assemble cells into 3D multicellular architectures. Overall, this
approach captures the dynamic and reversible nature of cell–cell interactions
and controls them noninvasively and sustainably both in space and time. This
provides a new way of studying cell–cell interactions and assembling cellular
building blocks into tissues with unmatched flexibility.
Intensiometric biosensors visualize the activity of multiple small GTPases in vivo.
Ras and Rho small GTPases are critical for numerous cellular processes including cell division, migration, and intercellular communication. Despite extensive efforts to visualize the spatiotemporal activity of these proteins, achieving the sensitivity and dynamic range necessary for in vivo application has been challenging. Here, we present highly sensitive intensiometric small GTPase biosensors visualizing the activity of multiple small GTPases in single cells in vivo. Red-shifted sensors combined with blue light-controllable optogenetic modules achieved simultaneous monitoring and manipulation of protein activities in a highly spatiotemporal manner. Our biosensors revealed spatial dynamics of Cdc42 and Ras activities upon structural plasticity of single dendritic spines, as well as a broad range of subcellular Ras activities in the brains of freely behaving mice. Thus, these intensiometric small GTPase sensors enable the spatiotemporal dissection of complex protein signaling networks in live animals.
Independent Control over Multiple Cell Types in Space and Time Using Orthogonal Blue and Red Light Switchable Cell Interactions.
Independent control over multiple cell–material interactions with high spatiotemporal resolution is a key for many biomedical applications and understanding cell biology, as different cell types can perform different tasks in a multicellular context. In this study, the binding of two different cell types to materials is orthogonally controlled with blue and red light providing independent regulation in space and time. Cells expressing the photoswitchable protein cryptochrome 2 (CRY2) on cell surface bind to N‐truncated CRY‐interacting basic helix–loop–helix protein 1 (CIBN)‐immobilized substrates under blue light and cells expressing the photoswitchable protein phytochrome B (PhyB ) on cell surface bind to phytochrome interaction factor 6 (PIF6)‐immobilized substrates under red light, respectively. These light‐switchable cell interactions provide orthogonal and noninvasive control using two wavelengths of visible light. Moreover, both cell–material interactions are dynamically switched on under light and reversible in the dark. The specificity of the CRY2/CIBN and PhyB/PIF6 interactions and their response to different wavelengths of light allow selectively activating the binding of one cell type with blue and the other cell type with red light in the presence of the other cell type.
Optogenetic Control of Ras/Erk Signaling Using the Phy-PIF System.
The Ras/Erk signaling pathway plays a central role in diverse cellular processes ranging from development to immune cell activation to neural plasticity to cancer. In recent years, this pathway has been widely studied using live-cell fluorescent biosensors, revealing complex Erk dynamics that arise in many cellular contexts. Yet despite these high-resolution tools for measurement, the field has lacked analogous tools for control over Ras/Erk signaling in live cells. Here, we provide detailed methods for one such tool based on the optical control of Ras activity, which we call "Opto-SOS." Expression of the Opto-SOS constructs can be coupled with a live-cell reporter of Erk activity to reveal highly quantitative input-to-output maps of the pathway. Detailed herein are protocols for expressing the Opto-SOS system in cultured cells, purifying the small molecule cofactor necessary for optical stimulation, imaging Erk responses using live-cell microscopy, and processing the imaging data to quantify Ras/Erk signaling dynamics.
Spatiotemporal control of gene expression by a light-switchable transgene system.
We developed a light-switchable transgene system based on a synthetic, genetically encoded light-switchable transactivator. The transactivator binds promoters upon blue-light exposure and rapidly initiates transcription of target transgenes in mammalian cells and in mice. This transgene system provides a robust and convenient way to spatiotemporally control gene expression and can be used to manipulate many biological processes in living systems with minimal perturbation.