Following an injury, a blood clot must form at the wound site to stop bleeding before skin repair can occur. Blood clots must satisfy a unique set of material requirements; they need to be sufficiently strong to resist pressure from the arterial blood flow but must be highly flexible to support large strains associated with tissue movement around the wound. These combined properties are enabled by a fibrous matrix consisting of the protein fibrin. Fibrin hydrogels can support large macroscopic strains owing to the unfolding transition of a-helical fibril structures to b sheets at the molecular level, among other reasons. Imaging protein secondary structure on the submicrometer length scale, we reveal that another length scale is relevant for fibrin function. We observe that the protein polymorphism in the gel becomes spatially heterogeneous on a micrometer length scale with increasing tensile strain, directly showing load-bearing inhomogeneity and nonaffinity. Supramolecular structural features in the hydrogel observed under strain indicate that a uniform fibrin hydrogel develops a composite-like microstructure in tension, even in the absence of cellular inclusions.

Accumulation of fat in muscle tissue as intramyocellular lipids (IMCLs) is closely related to the development of insulin resistance and subsequent type 2 diabetes. Most IMCLs organize into lipid droplets (LDs), the fates of which are regulated by lipid droplet coat proteins. Perilipin 5 (PLIN5) is an LD coating protein, which is strongly linked to lipid storage in muscle tissue. Here we employ a tandem in vitro/ex vivo approach and use chemical imaging by label-free, hyperspectral coherent Raman microscopy to quantify compositional changes in individual LDs upon PLIN5 overexpression. Our results directly show that PLIN5 overexpression in muscle alters individual LD composition and physiology, resulting in larger LDs with higher esterified acyl chain concentration, increased methylene content, and more saturated lipid species. These results suggest that lipotoxic protection afforded by natural PLIN5 upregulation in muscle involves molecular changes in lipid composition within LDs.

Collagen is the predominant protein in animal connective tissues and is widely used in tissue regeneration and other industrial applications. Marine organisms have gained interest as alternative, nonmammalian collagen sources for biomaterial applications because of potential medical and economic advantages. In this work, we present physicochemical and biofunctionality studies of acid solubilized collagen (ASC) from jellyfish Catostylus mosaicus (JASC), harvested from the Persian Gulf, compared with ASC from rat tail tendon (RASC), the industry-standard collagen used for biomedical research. From the protein subunit (alpha chain) pattern of JASC, we identified it as a type I collagen, and extensive molecular spectroscopic analyses showed similar triple helical molecular signatures for JASC and RASC. Atomic force microscopy of fibrillized JASC showed clear fibril reassembly upon pH neutralization though with different temperature and concentration dependence compared with RASC. Molecular (natively folded, nonfibrillized) JASC was shown to functionalize rigid substrates and promote MC3T3 preosteoblast cell attachment and proliferation better than RASC over 6 days. On blended collagen–agarose scaffolds, both RASC and JASC fibrils supported cell attachment and proliferation, and scaffolds with RASC fibrils showed more cell growth after 6 days compared with those scaffolds with JASC fibrils. These results demonstrate the potential for this new type I collagen as a possible alternative to mammalian type I collagen for biomaterial applications.

Cell-penetrating peptides (CPPs) are short peptide sequences that can translocate across cellular plasma membranes and are thus potential delivery vectors for diagnostic and therapeutic applications. Many CPPs exhibit some sort of structural polymorphism, where the secondary structure of the peptide is altered strongly by its local environment, which is believed to facilitate membrane translocation and uptake. However, much less is known about the fate and structure of CPPs within cells largely due to measurement difficulty. Here we employ isotopic labeling combined with hyperspectral, quantitative coherent Raman microscopy to localize a model CPP-penetratin-and determine its secondary structure in different cellular compartments. Our results show that penetratin is mostly α-helical in the cytosol and acquires a more β-sheet and random coil character in the nucleus. The increased helicity in the cytosol is similar to that seen in previous studies with model lipid membranes, suggesting that the peptide is associated with membranes in, e.g., endosomes (or lysosomes) in the cytosol. The ability to both localize and determine the secondary structure of a CPP within cells is critical for clarifying the mechanism of peptide-mediated translocation and delivery of cargo molecules to specific cellular destinations.