05. Encounters with the Deep

https://crossworks.holycross.edu/poetry2021/1004/thumbnail.jp

Single-Component Optogenetic Tools Fo Cytoskeletal Rearrangements

The Rho family of small GTPases coordinate actin cytoskeletal rearrangements underlying crucial cell processes including migration and mechanotransduction. Dysregulation in these signaling pathways has been associated with neurodegenerative disease and cancer. Rho GTPase signaling is tightly controlled in space and time: GTPases are activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase accelerating proteins (GAPs) at the plasma membrane. To study Rho GTPase signaling, several optogenetic tools have been developed, most of which use light to induce a protein-protein interaction, recruiting a GTPase-activating GEF to the plasma membrane. Other optogenetic strategies involve the use of single-chain photoswitches sterically occluding a constitutively active GTPase, which can result in undesirable high dark-state activity of the tool. We sought to create single-component optogenetic tools to perturb Rho GTPase signaling at the GTPase, GEF, and GAP level, resulting in lower dark state activity and easier implementation in mammalian systems.In this work, we used BcLOV4, a fungal photoreceptor which directly binds membrane lipids in response to blue light inputs, to recruit Rho signaling proteins to the membrane, resulting in spatiotemporally precise signaling perturbation. We created BcLOV4 activation tools using the GTPase and GEF from the three best studied Rho GTPase pathways: RhoA, which induces cell contraction through stress fiber formation; Rac1, which induces sheet-like lamellipodial protrusions; and Cdc42, which induces spiky filopodial protrusions. Notably, we demonstrated that the BcLOV4 system is compatible with wildtype GTPases, resulting in lower unintended pathway activation in the dark state. We also report progress toward the creation of RhoA termination tools using GAP domains and dominant-negative GTPases, allowing for the induction of signaling activation and termination on the same optogenetic platform. Using structural knowledge we gained from Rho GTPase tool development, we created a plasmid set and cloning workflow to simplify BcLOV4 tool engineering for other signaling targets. Together, the BcLOV4 optogenetic toolbox will further the study of Rho GTPase signaling and enable others to use this technology for single-component optogenetic membrane recruitment

Evolution of chemical diversity by coordinated gene swaps in type II polyketide gene clusters

Natural product biosynthetic pathways generate molecules of enormous structural complexity and exquisitely tuned biological activities. Studies of natural products have led to the discovery of many pharmaceutical agents, particularly antibiotics. Attempts to harness the catalytic prowess of biosynthetic enzyme systems, for both compound discovery and engineering, have been limited by a poor understanding of the evolution of the underlying gene clusters. We developed an approach to study the evolution of biosynthetic genes on a cluster-wide scale, integrating pairwise gene coevolution information with large-scale phylogenetic analysis. We used this method to infer the evolution of type II polyketide gene clusters, tracing the path of evolution from the single ancestor to those gene clusters surviving today. We identified 10 key gene types in these clusters, most of which were swapped in from existing cellular processes and subsequently specialized. The ancestral type II polyketide gene cluster likely comprised a core set of five genes, a roster that expanded and contracted throughout evolution. A key C24 ancestor diversified into major classes of longer and shorter chain length systems, from which a C20 ancestor gave rise to the majority of characterized type II polyketide antibiotics. Our findings reveal that (i) type II polyketide structure is predictable from its gene roster, (ii) only certain gene combinations are compatible, and (iii) gene swaps were likely a key to evolution of chemical diversity. The lessons learned about how natural selection drives polyketide chemical innovation can be applied to the rational design and guided discovery of chemicals with desired structures and properties

Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids

We report natural light–oxygen–voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ∼ 10−7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helixinthelinker regionbetween the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure–function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit

Realising the Olympic dream: vision, support and challenge

The sporting arena is replete with examples and anecdotes of great inspirational coaches that have led teams to success, often in the face of adversity and against seemingly better opponents. The role of the coach in developing and motivating athletes has also been the focus of much research in sport psychology (e.g., Challaduria 1990; Smith & Smoll, 2007). Despite the ease with which one readily accepts that coaches can be inspirational, the sport coaching literature is somewhat devoid of research on inspirational coaches and the effects of such coaches on athletic success. The purpose of the current paper is to theoretically delineate the inspirational effects of coaches in sport. Given the relative paucity of inspiration-related research in sport we draw upon contemporary theories of leadership from organisational and military psychology (e.g., transformational and charismatic leadership theories). We propose a sport-specific model of leadership that centres around the vision, support, and challenge meta-cognitive model developed by Arthur and Hardy in military contexts. The model posits that �great� coaches inspire their athletes by: (a) creating an inspirational vision of the future; (b) providing the necessary support to achieve the vision; and (c) providing the challenge to achieve the vision. The underlying proposition is that the vision provides meaning and direction for followers� effort. That is, the vision serves as the beacon around which all the sweat, pain and sacrifice involved in achieving success at the highest level in sport is directed. At the heart of this model is the notion that athletes can achieve their dreams provided they are inspired to do so; this is because all other things being equal the person who is motivated to practice longer and train harder will ultimately be the best. The current paper will delineate the coach�s role in inspiring the athlete to train harder and longer

Single-Component Optogenetic Tools Fo Cytoskeletal Rearrangements

The Rho family of small GTPases coordinate actin cytoskeletal rearrangements underlying crucial cell processes including migration and mechanotransduction. Dysregulation in these signaling pathways has been associated with neurodegenerative disease and cancer. Rho GTPase signaling is tightly controlled in space and time: GTPases are activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase accelerating proteins (GAPs) at the plasma membrane. To study Rho GTPase signaling, several optogenetic tools have been developed, most of which use light to induce a protein-protein interaction, recruiting a GTPase-activating GEF to the plasma membrane. Other optogenetic strategies involve the use of single-chain photoswitches sterically occluding a constitutively active GTPase, which can result in undesirable high dark-state activity of the tool. We sought to create single-component optogenetic tools to perturb Rho GTPase signaling at the GTPase, GEF, and GAP level, resulting in lower dark state activity and easier implementation in mammalian systems. In this work, we used BcLOV4, a fungal photoreceptor which directly binds membrane lipids in response to blue light inputs, to recruit Rho signaling proteins to the membrane, resulting in spatiotemporally precise signaling perturbation. We created BcLOV4 activation tools using the GTPase and GEF from the three best studied Rho GTPase pathways: RhoA, which induces cell contraction through stress fiber formation; Rac1, which induces sheet-like lamellipodial protrusions; and Cdc42, which induces spiky filopodial protrusions. Notably, we demonstrated that the BcLOV4 system is compatible with wildtype GTPases, resulting in lower unintended pathway activation in the dark state. We also report progress toward the creation of RhoA termination tools using GAP domains and dominant-negative GTPases, allowing for the induction of signaling activation and termination on the same optogenetic platform. Using structural knowledge we gained from Rho GTPase tool development, we created a plasmid set and cloning workflow to simplify BcLOV4 tool engineering for other signaling targets. Together, the BcLOV4 optogenetic toolbox will further the study of Rho GTPase signaling and enable others to use this technology for single-component optogenetic membrane recruitment

Optogenetics experiment checklist

Optogenetics, calcium imaging, and modern microscopy provide a powerful approach to uncover new biological phenomena and explore cellular and molecular mechanisms in ways that are both visual and quantitative. Paired properly, optogenetic approaches combined with calcium imaging can determine functional relationships within biological circuits. For example, stimulating or inhibiting neural populations, and subsequently recording neural activity, allows one to examine the presence of epistatic neural relationships. While technological advancement makes these methods more accessible, it should be emphasized that there are a number of understated complexities involved with these types of imaging-based experiments. Successful application of optogenetics requires good experimental design, controls, and proper understanding of microscopy limitations in order to generate clear and easily interpretable data. This is true both at the level of designing one's own experiments as well as for critically evaluating studies carried out by others. In a recent class we taught at Caltech, we introduced students to these concept, practical details, and limitations of optogenetic and calcium imaging experiments. Here we share a brief "checklist" from that class to make it easier for scientists to start their first hands-on optogenetic and calcium imaging experiments

The management of tension in organization : some preliminary findings

Series title handwritten on t.p.In: Industrial Management Review, Alfred P. Sloan School of Management, Massachusetts Institute of Technology, v. 6, no. 1, Fall, 1964