Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level

Scientific mobilization of keystone actors for biosphere stewardship

The biosphere crisis requires changes to existing business practices. We ask how corporations can become sustainability leaders, when constrained by multiple barriers to collaboration for biosphere stewardship. We describe how scientists motivated, inspired and engaged with ten of the world’s largest seafood companies, in a collaborative process aimed to enable science-based and systemic transformations (2015–2021). CEOs faced multiple industry crises in 2015 that incentivized novel approaches. New scientific insights, an invitation to collaborate, and a bold vision of transformative change towards ocean stewardship, created new opportunities and direction. Co-creation of solutions resulted in new knowledge and trust, a joint agenda for action, new capacities, international recognition, formalization of an organization, increased policy influence, time-bound goals, and convergence of corporate change. Independently funded scientists helped remove barriers to cooperation, provided means for reflection, and guided corporate strategies and actions toward ocean stewardship. By 2021, multiple individuals exercised leadership and the initiative had transitioned from preliminary and uncomfortable conversations, to a dynamic, operational organization, with capacity to perform global leadership in the seafood industry. Mobilizing transformational agency through learning, collaboration, and innovation represents a cultural evolution with potential to redirect and accelerate corporate action, to the benefit of business, people and the planet

Designed Fluorescent Probes Reveal Interactions between Amyloid-β(1–40) Peptides and GM1 Gangliosides in Micelles and Lipid Vesicles

A hallmark of the common Alzheimer's disease (AD) is the pathological conversion of its amphiphatic amyloid-β (Aβ) peptide into neurotoxic aggregates. In AD patients, these aggregates are often found to be tightly associated with neuronal GM1 ganglioside lipids, suggesting an involvement of GM1 not only in aggregate formation but also in neurotoxic events. Significant interactions were found between micelles made of newly synthesized fluorescent GM1 gangliosides labeled in the polar headgroup or the hydrophobic chain and Aβ(1–40) peptide labeled with a BODIPY-FL-C1 fluorophore at positions 12 and 26, respectively. From an analysis of energy transfer between the different fluorescence labels and their location in the molecules, we were able to place the Aβ peptide inside GM1 micelles, close to the hydrophobic-hydrophilic interface. Large unilamellar vesicles composed of a raftlike GM1/bSM/cholesterol lipid composition doped with labeled GM1 at various positions also interact with labeled Aβ peptide tagged to amino acids 2 or 26. A faster energy transfer was observed from the Aβ peptide to bilayers doped with 581/591-BODIPY-C11-GM1 in the nonpolar part of the lipid compared with 581/591-BODIPY-C5-GM1 residing in the polar headgroup. These data are compatible with a clustering process of GM1 molecules, an effect that not only increases the Aβ peptide affinity, but also causes a pronounced Aβ peptide penetration deeper into the lipid membrane; all these factors are potentially involved in Aβ peptide aggregate formation due to an altered ganglioside metabolism found in AD patients