Here we use time-lapse microscopy to analyse cell-matrix adhesions in cells expressing one of two different cytoskeletal proteins, paxillin or tensin, tagged with green fluorescent protein (GFP). Use of GFP-paxillin to analyse focal contacts and GFP-tensin to study fibrillar adhesions reveals that both types of major adhesion are highly dynamic. Small focal contacts often translocate, by extending centripetally and contracting peripherally, at a mean rate of 19 micrometers per hour. Fibrillar adhesions arise from the medial ends of stationary focal contacts, contain alpha5beta1 integrin and tensin but not other focal-contact components, and associate with fibronectin fibrils. Fibrillar adhesions translocate centripetally at a mean rate of 18 micrometers per hour in an actomyosin-dependent manner. We propose a dynamic model for the regulation of cell-matrix adhesions and for transitions between focal contacts and fibrillar adhesions, with the ability of the matrix to deform functioning as a mechanical switch.

As a central element of the extracellular matrix, collagen is intimately involved in tissue development, remodeling, and repair and confers high tensile strength to tissues. Numerous medical applications, particularly, wound healing, cell therapy, bone reconstruction, and cosmetic technologies, rely on its supportive and healing qualities. Its synthesis and assembly require a multitude of genes and post-translational modifications, where even minor deviations can be deleterious or even fatal. Historically, collagen was always extracted from animal and human cadaver sources, but bare risk of contamination and allergenicity and was subjected to harsh purification conditions resulting in irreversible modifications impeding its biofunctionality. In parallel, the highly complex and stringent post-translational processing of collagen, prerequisite of its viability and proper functioning, sets significant limitations on recombinant expression systems. A tobacco plant expression platform has been recruited to effectively express human collagen, along with three modifying enzymes, critical to collagen maturation. The plant extracted recombinant human collagen type I forms thermally stable helical structures, fibrillates, and demonstrates bioactivity resembling that of native collagen. Deployment of the highly versatile plant-based biofactory can be leveraged toward mass, rapid, and low-cost production of a wide variety of recombinant proteins. As in the case of collagen, proper planning can bypass plant-related limitations, to yield products structurally and functionally identical to their native counterparts.

Reactive oxygen species (ROS) comprise a group of noxious byproducts of oxidative processes which participate in the induction of many common diseases. However, understanding their role in the regulation of normal physiological redox signaling is currently evolving. Detailed study of the dynamic functions of ROS within the biological milieu is difficult because of their high chemical reactivity, short lifetime, minute concentrations and cytotoxicity at high concentrations. In this study, we show that increasing intracellular ROS levels, set off by controlled in situ photogeneration of a nontoxic bacteriochlorophyll-based sensitizer initiate responses in cultured melanoma cells. Using hydroethidine as detector, we determined light-dependent generation of superoxide and hydroxyl radicals in cell-free and cell culture models. Monitoring the ROS-induced responses revealed individual and differential behavior of protein kinases [p38, mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and Akt] as well as effects on the subcellular distribution of phosphorylated p38. Furthermore, alterations in morphology and motility and effects on cell viability as a function of time and photosensitizer doses were observed. Following mild ROS challenge, enzymatic and cellular changes were observed in the majority of the cells, without inducing extensive cell death. However, upon vigorous ROS challenge, a similar profile of the overall responses was observed, terminating in cell death. This study shows that precisely controlled photogeneration of ROS can provide simple, fine-tuned, noninvasive manipulation of ROS-sensitive cellular responses ranging from individual enzymes to gross behavior of target cells. The observations made with this tool enable a dynamic and causal correlation, presenting a new alternative for studying the role of ROS in cellular redox signaling.

Consequential to its essential role as a mechanical support and affinity regulator in extracellular matrices, collagen constitutes a highly sought after scaffolding material for regeneration and healing applications. However, substantiated concerns have been raised with regard to quality and safety of animal tissue-extracted collagen, particularly in relation to its immunogenicity, risk of disease transmission and overall quality and consistency. In parallel, contamination with undesirable cellular factors can significantly impair its bioactivity, vis-a-vis its impact on cell recruitment, proliferation and differentiation. High-scale production of recombinant human collagen Type I (rhCOL1) in the tobacco plant provides a source of an homogenic, heterotrimeric, thermally stable "virgin" collagen which self assembles to fine homogenous fibrils displaying intact binding sites and has been applied to form numerous functional scaffolds for tissue engineering and regenerative medicine. In addition, rhCOL1 can form liquid crystal structures, yielding a well-organized and mechanically strong membrane, two properties indispensable to extracellular matrix (ECM) mimicry. Overall, the shortcomings of animal- and cadaver-derived collagens arising from their source diversity and recycled nature are fully overcome in the plant setting, constituting a collagen source ideal for tissue engineering and regenerative medicine applications.

Collagen is a key component of the extracellular matrix, and by far the most prominent constituent of all load-bearing tissues. Its abundance and self-assembly capacities render it a practical scaffold material for tissue repair and regeneration applications. However, some difficulties exist in artificially regenerating functional collagen structures to match native tissues and their respective performances. There are two major limitations of existing collagen-based scaffolds: The first one is poor mechanical performance, and the second one is the failure to closely mimic natural tissues as to provide the necessary topographic and mechanical cues required for cell propagation and differentiation. The complexity of inducing sufficient order and alignment stands at the base of the impediments to successful formation of artificial collagen scaffolds, which closely match native tissue strength and morphology. Recombinant human collagen produced in transgenic tobacco plants has the capacity of forming highly concentrated liquid crystalline dope that can be aligned by application of shear force. Leveraging shear alignment of liquid crystalline recombinant human collagen opens new possibilities toward obtaining scaffolds that may be able to provide the necessary mechanical support, while closely mimicking the molecular signals and mechanical cues displayed to natural cell milieu. Such scaffolds may prove advantageous in the development of improved medical devices in fields, such as ophthalmology, neurology, and orthopedics.