RESUMEN
Fluorescent biosensors are valuable tools to monitor protein activities and the functional state of organelles in live cells. However, the information provided by fluorescent microscopy (FM) is mostly limited in resolution and lacks ultrastructural context information. Protein activities are confined to organelle zones with a distinct membrane morphology, which can only be seen by electron microscopy (EM). EM, however, intrinsically lacks information on protein activities. The lack of methods to integrate these two imaging modalities has hampered understanding the functional organization of cellular organelles. Here we introduce "functional correlative microscopy" (functional CLEM) to directly infer functional information from live cells to EM with nanometer resolution. We label and visualize live cells with fluorescent biosensors after which they are processed for EM and imaged using a volume electron microscopy technique. Within a single dataset we correlate hundreds of fluorescent spots enabling quantitative analysis of the functional-ultrastructural data. We employ our method to monitor essential functional parameters of late endo-lysosomal compartments, i.e., pH, calcium, enzyme activities and cholesterol content. Our data reveal a steep functional difference in enzyme activity between late endosomes and lysosomes and unexpectedly high calcium levels in late endosomes. The presented CLEM workflow is compatible with a large repertoire of probes and paves the way for large scale functional studies of all types of cellular structures.
Asunto(s)
Calcio , Microscopía Electrónica de Volumen , Humanos , Células HeLa , Microscopía Electrónica , LisosomasRESUMEN
Hydrogel-based 3D cell cultures are an emerging strategy for the regeneration of cartilage. In an attempt to regenerate dysfunctional intervertebral discs, nucleus pulposus (NP) cells can be cultured in hydrogels of various kinds and physical properties. Stiffness sensing through focal adhesions is believed to direct chondrogenesis, but the mechanisms by which this works are largely unknown. In this study we compared focal adhesion formation and glycosaminoglycan (GAG) deposition by NP cells in a range of hydrogels. Using a focal adhesion kinase (FAK) inhibitor, we demonstrated that focal adhesion signaling is involved in the response of NP cells in hydrogels that contain integrin binding sites (i.e. methacrylated gelatin (gelMA) and type II collagen), but not in hydrogels deplete from integrin binding sites such as alginate and agarose, or CD44-binding hydrogels based on hyaluronic acid. As a result of FAK inhibition we observedenhanced proteoglycan production in gelMA, but decreased production in type II collagen hydrogels, which could be explained by alteration in cell fate as supported by the increase in the adipogenic marker peroxisome proliferator-activated receptor gamma (PPARy). Furthermore, GAG deposition was inversely proportional to polymer concentration in integrin-binding gelMA, while no direct relationship was found for the non-integrin binding gels alginate and agarose. This corroborates our finding that focal adhesion formation plays an important role in NP cell response to its surrounding matrix. STATEMENT OF SIGNIFICANCE: Biomaterials are increasingly being investigated for regenerative medicine applications, including regeneration of the nucleus pulposus. Cells interact with their environment and are influenced by extracellular matrix or polymer properties. Insight in these interactions can improve regeneration and helps to understand degeneration processes. The role of focal adhesion formation in the regenerative response of nucleus pulposus cells is largely unknown. Therefore, the relation between materials, stiffness and focal adhesion formation is studied here.