Optogenetic activation of intracellular signaling based on light-inducible protein-protein homo-interactions

Dynamic protein-protein interactions are essential for proper cell functioning.Homointeraction events—physical interactions between the same type of proteins—represent a pivotal subset of protein-protein interactions that are widely exploited in activating intracellular signaling pathways.Capacities of modulating protein-protein interactions with spatial and temporal resolution are greatly desired to decipher the dynamic nature of signal transduction mechanisms.The emerging optogenetic technology,based on genetically encoded light-sensitive proteins,provides promising opportunities to dissect the highly complex signaling networks with unmatched specificity and spatiotemporal precision.Here we review recent achievements in the development of optogenetic tools enabling light-inducible protein-protein homo-interactions and their applications in optical activation of signaling pathways.

Engineering microparticles based on solidified stem cell secretome with an augmented pro-angiogenic factor portfolio for therapeutic angiogenesis

Tissue (re)vascularization strategies face various challenges, as therapeutic cells do not survive long enough in situ, while the administration of pro-angiogenic factors is hampered by fast clearance and insufficient ability to emulate complex spatiotemporal signaling. Here, we propose to address these limitations by engineering a functional biomaterial capable of capturing and concentrating the pro-angiogenic activities of mesenchymal stem cells (MSCs). In particular, dextran sulfate, a high molecular weight sulfated glucose polymer, supplemented to MSC cul-tures, interacts with MSC-derived extracellular matrix (ECM) components and facilitates their co-assembly and accumulation in the pericellular space. Upon decellularization, the resulting dextran sulfate-ECM hybrid material can be processed into MIcroparticles of SOlidified Secretome (MIPSOS). The insoluble format of MIPSOS protects protein components from degradation, while facilitating their sustained release. Proteomic analysis demonstrates that MIPSOS are highly enriched in pro-angiogenic factors, resulting in an enhanced pro-angiogenic bioactivity when compared to naïve MSC-derived ECM (cECM). Consequently, intravital microscopy of full-thickness skin wounds treated with MIPSOS demonstrates accelerated revascularization and healing, far superior to the ther-apeutic potential of cECM. Hence, the microparticle-based solidified stem cell secretome provides a promising platform to address major limitations of current therapeutic angiogenesis approaches.

Demarcating the membrane damage for the extraction of functional mitochondria

Defective mitochondria have been linked to several critical human diseases such as neurodegenerative disorders,cancers and cardiovascular disease.However,the detailed characterization of mitochondria has remained relatively unexplored,largely due to the lack of effective extraction methods that may sufficiently retain the functionality of mitochondria,particularly when limited amount of sample is considered.In this study,we explore the possibility of modulating hydrodynamic stress through a cross-junction geometry at microscale to selectively disrupt the cellular membrane while mitochondrial membrane is secured.The operational conditions are empirically optimized to effectively shred the cell membranes while keeping mitochondria intact for the model mammalian cell lines,namely human embryonic kidney cells,mouse muscle cells and neuroblastoma cells.Unsurprisingly,the disruption of cell membranes with higher elastic moduli(neuroblastoma)requires elevated stress.This study also presents a comparative analysis of total protein yield and concentrations of extracted functional mitochondria with two commercially available mitochondria extraction approaches,the Dounce Homogenizer and the Qproteome®Mitochondria Isolation Kit,in a range of cell concentrations.Our findings show that the proposed“microscale cell shredder”yields at least 40%more functional mitochondria than the two other approaches and is able to preserve the morphological integrity of extracted mitochondria,particularly at low cell concentrations(5–20×10^(4) cells/mL).Characterized by its capability of rapidly processing a limited quantity of samples(200μL),demarcating the membrane damage through the proposed microscale cell shredder represents a novel strategy to extract subcellular organelles from clinical samples.

High spatial and temporal resolution synthetic aperture phase microscopy

A new optical microscopy technique,termed high spatial and temporal resolution synthetic aperture phase microscopy(HISTR-SAPM),is proposed to improve the lateral resolution of wide-field coherent imaging.Under plane wave illumination,the resolution is increased by twofold to around 260 nm,while achieving millisecond-level temporal resolution.In HISTR-SAPM,digital micromirror devices are used to actively change the sample illumination beam angle at high speed with high stability.An off-axis interferometer is used to measure the sample scattered complex fields,which are then processed to reconstruct high-resolution phase images.Using HISTR-SAPM,we are able to map the height profiles of subwavelength photonic structures and resolve the period structures that have 198 nm linewidth and 132 nm gap(i.e.,a full pitch of 330 nm).As the reconstruction averages out laser speckle noise while maintaining high temporal resolution,HISTR-SAPM further enables imaging and quantification of nanoscale dynamics of live cells,such as red blood cell membrane fluctuations and subcellular structure dynamics within nucleated cells.We envision that HISTR-SAPM will broadly benefit research in material science and biology.

Portable quantitative phase microscope for material metrology and biological imaging

Quantitative phase microscopy(QPM)has emerged as an important tool for material metrology and biological imaging.For broader adoption in those applications,we have proposed and demonstrated a new portable off-axis QPM method,which works in both transmission and reflection modes to meet different sample measurement requirements.The temporal and spatial sensitivities of our system,as quantified by optical path-length difference values,are 0.65 nm and 1.04 nm,respectively.To demonstrate its applicability for a wide range of applications,we deployed our system for profiling transistor gold electrode samples,observing red blood cell membrane fluctuations,imaging living cells flowing in a microfluidic chip,etc.Our portable QPM system has a low-cost design and involves a simple and robust phase-retrieval algorithm that we envision will allow for broader deployment at different environmental settings,including in resource-limited sites and integration with other metrology or imaging modalities.