Adaptation to Stressors by Systemic Amyloidogenesis

Cells facing environmental threats have developed numerous coping mechanisms, and HypOxystation users Tim Audas and Stephen Lee have uncovered a fascinating new cellular strategy to remain viable under stress and restore homeostasis when the crisis ends. In their recent paper “Adaptation to Stressors by Systemic Protein Amyloidogenesis”, they describe a physiological process of amyloidogenesis which cells activate under stress conditions, such as hypoxia and acidosis, to remove copious amounts of heterogeneous proteins from circulation, enabling cells to survive in a dormant state. This discovery expands our current view of amyloids as a rare and pathological phenomenon associated with neuropathies such as Alzheimer’s and Parkinson’s diseases, and exposes a novel post-translational, regulatory form of protein organization.

Using a combination of Congo red staining, proteinase K digestion, and OC antibody detection on cells exposed to a variety of stimuli, Audas et al. were able to identify nuclear foci consisting of immobilized, insoluble protein in a crossed β-sheet conformation which they named A-Bodies. In amyloidogenic proteins such as VHL and RNF8, an ACM (amyloid-converting motif) containing arginine and histidine was identified as essential for capture specifically in the A-bodies; a similar motif was also identified in the pathological β-amyloid associated with Alzheimer’s disease. Upon environmental insult, the ACM interacts with ribosomal intergenic spacer RNA (rIGSRNA) to concentrate the proteins and trigger their polymerization in the A-bodies allowing the cells to enter a dormant state.

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Natural amyloid aggregation is induced by cellular stress

Cellular ability to react to stressors such as extreme temperatures, hypoxia, determines the sustainability of the entire organism. Tim Audas at Simon Fraser University has uncovered the mechanism by which proteins are reversibly folded and immobilized in amyloid bodies in response to stress. In a presentation he gave at the Keystone Symposium on “Cellular Stress Responses and Infectious Agents” in Santa Fe, Tim Audas describes this process of amyloidogenesis as a means of ensuring survival and homeostasis under adverse conditions. Amyloidogenesis, which is well-known in the context of neurological disorders such as Alzheimer’s and Parkinson’s disease, helps the cell to enter a state of protective dormancy by sequestering diverse proteins in sub-nuclear foci, termed A-bodies. To mimic physiological stress, Dr. Audas cultured cells at 1% oxygen in the HypOxystation, also exposing them to acidosis for additional stress. The HypOxystation’s closed workstation format ensures controlled oxygen, CO2, temperature and humidity conditions throughout the duration of culture and manipulation. Dr. Audas states that, “The workstation is critical because it allows us to manipulate the cells within a low oxygen environment and, unlike an incubator, maintains the O2 levels when cells are taken in and out, avoiding spikes of normoxia“.

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Sequestration in Speckles

The HIF (hypoxia-inducible factor) family of oxygen-sensing proteins are a crucial element of cells’ responses to alterations in their immediate environment, kicking off a signaling cascade involving more than 1000 genes. HypOxystation users Taylor and See at the University of Liverpool describe novel insights into the subcellular localization of some of the HIF proteins and why the “where” determines the “how”.

HIF-2α and HIF-1α both form heterodimers with HIF-1β, and while similarities abound between the isoforms, the two subunits are differentially expressed and regulated and have distinctly separate target genes. Taylor and See triggered HIF activation using microscope stage incubators and the Hypoxystation by Don Whitley Scientific to incubate HeLa cells in hypoxia (1%). They found that while HIF-1α distributes homogenously in the nucleus, HIF-2α diffuses freely through the nucleus but is concentrated in speckles that are tethered to nuclear structures close to active RNA polymerases. This distribution is not significantly altered by low oxygen levels.

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TC-3 and TEB Bioreactors provide physiological conditions for cell culture

The TC-3 and TEB bioreactors are being used in numerous labs to provide physiological conditions for the culture of blood vessels, cardiac muscle, bone, cartilage, ligaments, tendons and skin. Here, we would like to introduce some of the research being published by these labs. 

I. A recent paper published by TC-3 users Hillary et al. (“Developing Repair Materials for Stress Urinary Incontinence to Withstand Dynamic Distension” 2016; PLoS ONE 11(3):e0149971. doi:10.1371/journal.pone.0149971) examines options for culturing adipose derived stem cells (ADSC) for use in surgical repair meshes.  Using the TC-3 to generate cyclic uniaxial distension, Hillary’s lab compared the current standard scaffold material polypropylene with Poly-L-lactic acid ((PLA) and polyurethanes (PU), focusing specifically on their supportive properties and biocompatibility, as measured by cell attachment, proliferation, and matrix production. They found that prolonged mechanical distension in vitro caused polypropylene to fail, while a combination of PLA with PU greatly improved dynamic distension and cell interaction properties of the mesh. The authors conclude that “the key finding of this study is that subjecting materials in vitro to dynamic strain reveals significant changes in their mechanical properties after only 7 days. … We suggest that this dynamic assessment is crucial in the development of materials for use in the pelvic floor.”

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Avoiding Spikes of Normoxia

“Using the HypOxystation is critical to our work because… unlike an incubator, it maintains the O2 levels when cells are taken in and out, avoiding spikes of normoxia.” Timothy Audas, University of Miami.

Responses to physiological cues are governed on a cellular level by changes in protein levels, and HypOxystation users Ho et al. describe their research on mechanisms mediating these responses in their newest paper "Systemic Reprogramming of Translation Efficiencies on Oxygen Stimulus”  (Cell Reports 14, 1293-1300, 2016).  Contrary to the widely held belief that transcription and mRNA levels are the main regulators of protein expression, Dr. Ho and Dr. Timothy Audas assert that translation efficiency Te of mRNA’s that are already present in the cell exerts a much larger influence than previously thought, especially in response to a stimulus such as hypoxia.

Glioblastoma and renal clear cell carcinoma cells were maintained at hypoxia (1%) in the H35 HypOxystation and at normoxia, and mRNA levels and protein expression were compared. The correlation between mRNA and protein output was quite weak, leading the authors to postulate that cellular response to oxygen stimulus occurs through a switch in Te rather than in transcription levels. Gene silencing of hypoxic and normoxic cell cultures via transient transfection with siRNA confirmed the existence of an alternative translation initiation complex binding to the 5’ cap of the mRNA, termed hypoxic eIF4F (eIF4FH), that is not in use at normoxia. The HypOxystation reliably creates physiological conditions for cells habituated to much lower oxygen than the ambient 21%, while also controlling CO2, temperature and humidity. Working inside the HypOxystation via gloveless sleeves allows users to culture and manipulate cells for extended periods of time, without ever exposing them to the “oxygen shock” of the lab atmosphere. In an interview with the University of Guelph, co-author and HypOxystation user Jim Uniacke describes using the HypOxystation to design a suicide mRNA strategy targeting hypoxic tumors.

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