Investigations of human myosin VI targeting using optogenetically controlled cargo loading

Abstract

Myosins play countless critical roles in the cell, each requiring it to be activated at a specific location and time. To control myosin VI with this specificity, we created an optogenetic tool for activating myosin VI by fusing the light-sensitive Avena sativa phototropin1 LOV2 domain to a peptide from Dab2 (LOVDab), a myosin VI cargo protein. Our approach harnesses the native targeting and activation mechanism of myosin VI, allowing direct inferences on myosin VI function. LOVDab robustly recruits human full-length myosin VI to various organelles in vivo and hinders peroxisome motion in a lightcontrollable manner. LOVDab also activates myosin VI in an in vitro gliding filament assay. Our data suggest that protein and lipid cargoes cooperate to activate myosin VI, allowing myosin VI to integrate Ca 2+, lipid, and protein cargo signals in the cell to deploy in a site-specific manner.otor proteins play countless roles in biology, each requiring the motor to be recruited and activated at a particular time and place inside the cell. To dissect these multiple roles, we must develop tools that allow us to control the recruitment and activation process. One promising technique for achieving this goal is through optogenetics (1). Optogenetics involves the engineering and application of optically controlled, genetically encoded proteins and is transforming the fields of neuro-and cell biology (2, 3). A major benefit of optogenetics is that proteins are activated using light, which allows for high temporal and spatial control over a protein of interest.Myosin VI is a motor protein whose study could particularly benefit from optogenetic control. It is the only myosin known to walk toward the pointed end of actin filaments (4, 5). This property enables it to perform a diverse array of cellular functions, including cell division, endosome trafficking, autophagy, and Golgi and plasma membrane anchoring (6-9). Myosin VI is also autoinhibited, a property that is commonly found in other myosins (10). When myosin VI binds to cargo through specialized adaptor proteins, this autoinhibition is relieved through a poorly understood mechanism likely involving the disruption of an interaction between its cargo binding domain (CBD) and the myosin head (11,12). Dissociation of the CBD from the head both frees the head to bind tightly to actin and exposes dimerization sites throughout the tail domain of myosin VI, allowing it to become a processive dimer (13-15). In some cases, myosin VI could conceivably function as a monomer, for example when fulfilling its role as a membrane tether during spermatid individualization (16). If this is the case, more work is needed to elucidate the cellular signals that determine its oligomeric state at each site of action.Myosin VI has two classes of cargo proteins that bind to distinct, conserved motifs on the myosin VI C terminus (17). Disabled2, or Dab2, belongs to the class of cargo proteins that bind to a conserved WWY site on myosin (18). Optineurin (OPTN) is a member of the second class that binds ...

Engaging myosin VI tunes motility, morphology and identity in endocytosis

“…To overcome this, our fusion proteins were adapted to use the FKBP‐FRB rapamycin‐inducible dimerization system in place of optogenetic triggering (Figure A). To test the efficacy of this system, we first utilized this system with peroxisomes (a non‐native, but stable and tractable organelle) as cargo, as has been reported by other groups . Labeling of peroxisomes was observed within 1 hour of rapamycin (100 nM) addition (Figure A,B).…”

“…However, because of the dynamic nature of the endocytic pathway, investigating factors that influence organelle homeostasis requires spatial and temporal control over motor activity that cannot be assessed from the complete knockdown of either motor or its cargo adaptor. Several studies have used a combination of chemical dimerization and optogenetic approaches to target motor proteins to subcellular organelles . In this study, we have leveraged these approaches to probe the functional outcomes of myosin VI engagement in the endocytic pathway.…”

“…This study demonstrates that the synthetic engagement of myosin VI motility on endosomes can influence their functional identity. While previous studies have demonstrated a link between Rab5 activity and microtubule‐based motor activity, this study links observations on myosin motility in cells with the functional consequences of myosin engagement …”

“…The first utilizes the formation of chemically inducible dimers between FKBP and FRB upon the addition of rapamycin or rapalogs . The second leverages the well‐established optogenetic pairs of (frequently, ePDZ1b and LOV2—though others have been used) to trigger reversible dimerization using light stimulation . These techniques have been extensively used to control organelle positioning and explore actin/microtubule motor competition.…”

An optimized toolbox for the optogenetic control of intracellular transport

“…To directly control organelle transport, we and others have developed assays using induced heterodimerization of organelle adaptor proteins to specific molecular motors (Adrian et al, 2017; Ballister et al, 2015; Duan et al, 2015; French et al, 2017; Gutnick et al, 2019; Harterink et al, 2016; Hoogenraad et al, 2003; Janssen et al, 2017; Kapitein et al, 2010a; Kapitein et al, 2010b; van Bergeijk et al, 2015). Inducing selective binding of motor proteins to specific organelles mediates directed transport along the cytoskeleton, which allows the selective subcellular enrichment or depletion of organelles.…”