A high affinity monoclonal antibody against HLA-E-VL9 enhances natural killer cell anti-tumor killing.

Natural killer (NK) cells kill target cells following triggering via germline-encoded receptors interacting with target cell-expressed ligands (direct killing), or via antibody-dependent cellular cytotoxicity (ADCC) mediated by FcγRIIIa. NK cytotoxicity is modulated by signaling through activating or inhibitory receptors. A major checkpoint is mediated by the NK inhibitory receptor NKG2A/CD94 and its target cell ligand, HLA-E, which is complexed with HLA signal sequence-derived peptides termed VL9 (HLA-E-VL9). We have previously reported the isolation of a murine HLA-E-VL9-specific IgM antibody 3H4 and the generation of a higher affinity IgG version (3H4v3). Here we have used phage display library selection to generate a high affinity version of 3H4v3, called 3H4v31, with an ∼700 fold increase in binding affinity. We show using an HLA-E-VL9+ K562 tumor model that, in vitro, the addition of 3H4v31 to target cells increased direct killing of targets by CD16-negative NK cell line NK-92 and also mediated ADCC by NK-92 cells transfected with CD16. Moreover, ADCC by primary NK cells was also enhanced in vitro by 3H4v31. 3H4v31 was also able to bind and enhance target cell lysis of endogenously expressed HLA-E-VL9 on human cervical cancer and human pancreatic cancer cell lines. In vivo, 3H4v31 slowed the growth rate of HLA-E-VL9+ K562 tumors implanted into NOD/SCID/IL2rγ null mice compared to isotype control when injected with NK-92 cells intratumorally. Together, these data demonstrate that mAb 3H4v31 can enhance NK cell killing of HLA-E-VL9-expressing tumor cells in vitro by both direct killing activity and by ADCC. Moreover, mAb 3H4v31 can enhance NK cell control of tumor growth in vivo. We thus identify HLA-E-VL9 monoclonal antibodies as a promising novel anti-tumor immunotherapy. One Sentence Summary: A high affinity monoclonal antibody against HLA-E-VL9 enhances natural killer cell anti-tumor killing by checkpoint inhibition and antibody dependent cellular cytotoxicity.

Engineered immunogens to elicit antibodies against conserved coronavirus epitopes.

Immune responses to SARS-CoV-2 primarily target the receptor binding domain of the spike protein, which continually mutates to escape acquired immunity. Other regions in the spike S2 subunit, such as the stem helix and the segment encompassing residues 815-823 adjacent to the fusion peptide, are highly conserved across sarbecoviruses and are recognized by broadly reactive antibodies, providing hope that vaccines targeting these epitopes could offer protection against both current and emergent viruses. Here we employ computational modeling to design scaffolded immunogens that display the spike 815-823 peptide and the stem helix epitopes without the distracting and immunodominant receptor binding domain. These engineered proteins bind with high affinity and specificity to the mature and germline versions of previously identified broadly protective human antibodies. Epitope scaffolds interact with both sera and isolated monoclonal antibodies with broadly reactivity from individuals with pre-existing SARS-CoV-2 immunity. When used as immunogens, epitope scaffolds elicit sera with broad betacoronavirus reactivity and protect as "boosts" against live virus challenge in mice, illustrating their potential as components of a future pancoronavirus vaccine.

Rho MultiBinder, a fluorescent biosensor that reports the activity of multiple GTPases.

Imaging two or more fluorescent biosensors in the same living cell can reveal the spatiotemporal coordination of protein activities. However, using multiple Förster resonance energy transfer (FRET) biosensors together is challenging due to toxicity and the need for orthogonal fluorophores. Here we generate a biosensor component that binds selectively to the activated conformation of three different proteins. This enabled multiplexed FRET with fewer fluorophores, and reduced toxicity. We generated this MultiBinder (MB) reagent for the GTPases RhoA, Rac1, and Cdc42 by combining portions of the downstream effector proteins Pak1 and Rhotekin. Using FRET between mCherry on the MB and YPet or mAmetrine on two target proteins, the activities of any pair of GTPases could be distinguished. The MB was used to image Rac1 and RhoA together with a third, dye-based biosensor for Cdc42. Quantifying effects of biosensor combinations on the frequency, duration, and velocity of cell protrusions and retractions demonstrated reduced toxicity. Multiplexed imaging revealed signaling hierarchies between the three proteins at the cell edge where they regulate motility.

Engineered Immunogens to Elicit Antibodies Against Conserved Coronavirus Epitopes.

Immune responses to SARS-CoV-2 primarily target the receptor binding domain of the spike protein, which continually mutates to escape acquired immunity. Other regions in the spike S2 subunit, such as the stem helix and the segment encompassing residues 815-823 adjacent to the fusion peptide, are highly conserved across sarbecoviruses and are recognized by broadly reactive antibodies, providing hope that vaccines targeting these epitopes could offer protection against both current and emergent viruses. Here we employed computational modeling to design scaffolded immunogens that display the spike 815-823 peptide and the stem helix epitopes without the distracting and immunodominant RBD. These engineered proteins bound with high affinity and specificity to the mature and germline versions of previously identified broadly protective human antibodies. Epitope scaffolds interacted with both sera and isolated monoclonal antibodies with broadly reactivity from individuals with pre-existing SARS-CoV-2 immunity. When used as immunogens, epitope scaffolds elicited sera with broad betacoronavirus reactivity and protected as "boosts" against live virus challenge in mice, illustrating their potential as components of a future pancoronavirus vaccine.

Regulation of local GTP availability controls RAC1 activity and cell invasion.

Physiological changes in GTP levels in live cells have never been considered a regulatory step of RAC1 activation because intracellular GTP concentration (determined by chromatography or mass spectrometry) was shown to be substantially higher than the in vitro RAC1 GTP dissociation constant (RAC1-GTP Kd). Here, by combining genetically encoded GTP biosensors and a RAC1 activity biosensor, we demonstrated that GTP levels fluctuating around RAC1-GTP Kd correlated with changes in RAC1 activity in live cells. Furthermore, RAC1 co-localized in protrusions of invading cells with several guanylate metabolism enzymes, including rate-limiting inosine monophosphate dehydrogenase 2 (IMPDH2), which was partially due to direct RAC1-IMPDH2 interaction. Substitution of endogenous IMPDH2 with IMPDH2 mutants incapable of binding RAC1 did not affect total intracellular GTP levels but suppressed RAC1 activity. Targeting IMPDH2 away from the plasma membrane did not alter total intracellular GTP pools but decreased GTP levels in cell protrusions, RAC1 activity, and cell invasion. These data provide a mechanism of regulation of RAC1 activity by local GTP pools in live cells.

Correcting Artifacts in Ratiometric Biosensor Imaging; an Improved Approach for Dividing Noisy Signals.

The accuracy of biosensor ratio imaging is limited by signal/noise. Signals can be weak when biosensor concentrations must be limited to avoid cell perturbation. This can be especially problematic in imaging of low volume regions, e.g., along the cell edge. The cell edge is an important imaging target in studies of cell motility. We show how the division of fluorescence intensities with low signal-to-noise at the cell edge creates specific artifacts due to background subtraction and division by small numbers, and that simply improving the accuracy of background subtraction cannot address these issues. We propose a new approach where, rather than simply subtracting background from the numerator and denominator, we subtract a noise correction factor (NCF) from the numerator only. This NCF can be derived from the analysis of noise distribution in the background near the cell edge or from ratio measurements in the cell regions where signal-to-noise is high. We test the performance of the method first by examining two noninteracting fluorophores distributed evenly in cells. This generated a uniform ratio that could provide a ground truth. We then analyzed actual protein activities reported by a single chain biosensor for the guanine exchange factor (GEF) Asef, and a dual chain biosensor for the GTPase Cdc42. The reduction of edge artifacts revealed persistent Asef activity in a narrow band (∼640 nm wide) immediately adjacent to the cell edge. For Cdc42, the NCF method revealed an artifact that would have been obscured by traditional background subtraction approaches.

Author Correction: Force-exerting perpendicular lateral protrusions in fibroblastic cell contraction.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Force-exerting perpendicular lateral protrusions in fibroblastic cell contraction.

Aligned extracellular matrix fibers enable fibroblasts to undergo myofibroblastic activation and achieve elongated shapes. Activated fibroblasts are able to contract, perpetuating the alignment of these fibers. This poorly understood feedback process is critical in chronic fibrosis conditions, including cancer. Here, using fiber networks that serve as force sensors, we identify "3D perpendicular lateral protrusions" (3D-PLPs) that evolve from lateral cell extensions named twines. Twines originate from stratification of cyclic-actin waves traversing the cell and swing freely in 3D to engage neighboring fibers. Once engaged, a lamellum forms and extends multiple secondary twines, which fill in to form a sheet-like PLP, in a force-entailing process that transitions focal adhesions to activated (i.e., pathological) 3D-adhesions. The specific morphology of PLPs enables cells to increase contractility and force on parallel fibers. Controlling geometry of extracellular networks confirms that anisotropic fibrous environments support 3D-PLP formation and function, suggesting an explanation for cancer-associated desmoplastic expansion.

Engineering Optogenetic Protein Analogs.

This chapter provides an overview of the technologies we have developed to control proteins with light. First, we focus on the LOV domain, a versatile building block with reversible photo-response, kinetics tunable through mutagenesis, and ready expression in a broad range of cells and animals. Incorporation of LOV into proteins produced a variety of approaches: simple steric block of the active site released when irradiation lengthened a linker (PA-GTPases), reversible release from sequestration at mitochondria (LOVTRAP), and Z-lock, a method in which a light-cleavable bridge is placed where it occludes the active site. The latter two methods make use of Zdk, small engineered proteins that bind selectively to the dark state of LOV. In order to control endogenous proteins, inhibitory peptides are embedded in the LOV domain where they are exposed only upon irradiation (PKA and MLCK inhibition). Similarly, controlled exposure of a nuclear localization sequence and nuclear export sequence is used to reversibly send proteins into the nucleus. Another avenue of engineering makes use of the heterodimerization of FKBP and FRB proteins, induced by the small molecule rapamycin. We control rapamycin with light or simply add it to target cells. Incorporation of fused FKBP-FRB into kinases, guanine exchange factors, or GTPases leads to rapamycin-induced protein activation. Kinases are engineered so that they can interact with only a specific substrate upon activation. Recombination of split proteins using rapamycin-induced conformational changes minimizes spontaneous reassembly. Finally, we explore the insertion of LOV or rapamycin-responsive domains into proteins such that light-induced conformational changes exert allosteric control of the active site. We hope these design ideas will inspire new applications and broaden our reach towards dynamic biological processes that unfold when studied in vivo.

Multiplexed GTPase and GEF biosensor imaging enables network connectivity analysis.

Here we generate fluorescence resonance energy transfer biosensors for guanine exchange factors (GEFs) by inserting a fluorescent protein pair in a structural 'hinge' common to many GEFs. Fluorescent biosensors can map the activation of signaling molecules in space and time, but it has not been possible to quantify how different activation events affect one another or contribute to a specific cell behavior. By imaging the GEF biosensors in the same cells as red-shifted biosensors of Rho GTPases, we can apply partial correlation analysis to parse out the extent to which each GEF contributes to the activation of a specific GTPase in regulating cell movement. Through analysis of spontaneous cell protrusion events, we identify when and where the GEF Asef regulates the GTPases Cdc42 and Rac1 to control cell edge dynamics. This approach exemplifies a powerful means to elucidate the real-time connectivity of signal transduction networks.

Spatiotemporal dynamics of GEF-H1 activation controlled by microtubule- and Src-mediated pathways.

Rho family GTPases are activated with precise spatiotemporal control by guanine nucleotide exchange factors (GEFs). Guanine exchange factor H1 (GEF-H1), a RhoA activator, is thought to act as an integrator of microtubule (MT) and actin dynamics in diverse cell functions. Here we identify a GEF-H1 autoinhibitory sequence and exploit it to produce an activation biosensor to quantitatively probe the relationship between GEF-H1 conformational change, RhoA activity, and edge motion in migrating cells with micrometer- and second-scale resolution. Simultaneous imaging of MT dynamics and GEF-H1 activity revealed that autoinhibited GEF-H1 is localized to MTs, while MT depolymerization subadjacent to the cell cortex promotes GEF-H1 activation in an ~5-µm-wide peripheral band. GEF-H1 is further regulated by Src phosphorylation, activating GEF-H1 in a narrower band ~0-2 µm from the cell edge, in coordination with cell protrusions. This indicates a synergistic intersection between MT dynamics and Src signaling in RhoA activation through GEF-H1.

High Rac1 activity is functionally translated into cytosolic structures with unique nanoscale cytoskeletal architecture.

Rac1 activation is at the core of signaling pathways regulating polarized cell migration. So far, it has not been possible to directly explore the structural changes triggered by Rac1 activation at the molecular level. Here, through a multiscale imaging workflow that combines biosensor imaging of Rac1 dynamics with electron cryotomography, we identified, within the crowded environment of eukaryotic cells, a unique nanoscale architecture of a flexible, signal-dependent actin structure. In cell regions with high Rac1 activity, we found a structural regime that spans from the ventral membrane up to a height of ∼60 nm above that membrane, composed of directionally unaligned, densely packed actin filaments, most shorter than 150 nm. This unique Rac1-induced morphology is markedly different from the dendritic network architecture in which relatively short filaments emanate from existing, longer actin filaments. These Rac1-mediated scaffold assemblies are devoid of large macromolecules such as ribosomes or other filament types, which are abundant at the periphery and within the remainder of the imaged volumes. Cessation of Rac1 activity induces a complete and rapid structural transition, leading to the absence of detectable remnants of such structures within 150 s, providing direct structural evidence for rapid actin filament network turnover induced by GTPase signaling events. It is tempting to speculate that this highly dynamical nanoscaffold system is sensitive to local spatial cues, thus serving to support the formation of more complex actin filament architectures-such as those mandated by epithelial-mesenchymal transition, for example-or resetting the region by completely dissipating.

STEF/TIAM2-mediated Rac1 activity at the nuclear envelope regulates the perinuclear actin cap.

The perinuclear actin cap is an important cytoskeletal structure that regulates nuclear morphology and re-orientation during front-rear polarisation. The mechanisms regulating the actin cap are currently poorly understood. Here, we demonstrate that STEF/TIAM2, a Rac1 selective guanine nucleotide exchange factor, localises at the nuclear envelope, co-localising with the key perinuclear proteins Nesprin-2G and Non-muscle myosin IIB (NMMIIB), where it regulates perinuclear Rac1 activity. We show that STEF depletion reduces apical perinuclear actin cables (a phenotype rescued by targeting active Rac1 to the nuclear envelope), increases nuclear height and impairs nuclear re-orientation. STEF down-regulation also reduces perinuclear pMLC and decreases myosin-generated tension at the nuclear envelope, suggesting that STEF-mediated Rac1 activity regulates NMMIIB activity to promote stabilisation of the perinuclear actin cap. Finally, STEF depletion decreases nuclear stiffness and reduces expression of TAZ-regulated genes, indicating an alteration in mechanosensing pathways as a consequence of disruption of the actin cap.

Coordination by Cdc42 of Actin, Contractility, and Adhesion for Melanoblast Movement in Mouse Skin.

The individual molecular pathways downstream of Cdc42, Rac, and Rho GTPases are well documented, but we know surprisingly little about how these pathways are coordinated when cells move in a complex environment in vivo. In the developing embryo, melanoblasts originating from the neural crest must traverse the dermis to reach the epidermis of the skin and hair follicles. We previously established that Rac1 signals via Scar/WAVE and Arp2/3 to effect pseudopod extension and migration of melanoblasts in skin. Here we show that RhoA is redundant in the melanocyte lineage but that Cdc42 coordinates multiple motility systems independent of Rac1. Similar to Rac1 knockouts, Cdc42 null mice displayed a severe loss of pigmentation, and melanoblasts showed cell-cycle progression, migration, and cytokinesis defects. However, unlike Rac1 knockouts, Cdc42 null melanoblasts were elongated and displayed large, bulky pseudopods with dynamic actin bursts. Despite assuming an elongated shape usually associated with fast mesenchymal motility, Cdc42 knockout melanoblasts migrated slowly and inefficiently in the epidermis, with nearly static pseudopods. Although much of the basic actin machinery was intact, Cdc42 null cells lacked the ability to polarize their Golgi and coordinate motility systems for efficient movement. Loss of Cdc42 de-coupled three main systems: actin assembly via the formin FMNL2 and Arp2/3, active myosin-II localization, and integrin-based adhesion dynamics.

MRCK-1 Drives Apical Constriction in C. elegans by Linking Developmental Patterning to Force Generation.

Apical constriction is a change in cell shape that drives key morphogenetic events including gastrulation and neural tube formation. Apical force-producing actomyosin networks drive apical constriction by contracting while connected to cell-cell junctions. The mechanisms by which developmental patterning regulates these actomyosin networks and associated junctions with spatial precision are not fully understood. Here we identify a myosin light-chain kinase MRCK-1 as a key regulator of C. elegans gastrulation that integrates spatial and developmental patterning information. We show that MRCK-1 is required for activation of contractile actomyosin dynamics and elevated cortical tension in the apical cell cortex of endoderm precursor cells. MRCK-1 is apically localized by active Cdc42 at the external, cell-cell contact-free surfaces of apically constricting cells, downstream of cell fate determination mechanisms. We establish that the junctional components α-catenin, ß-catenin, and cadherin become highly enriched at the apical junctions of apically constricting cells and that MRCK-1 and myosin activity are required in vivo for this enrichment. Taken together, our results define mechanisms that position a myosin activator to a specific cell surface where it both locally increases cortical tension and locally enriches junctional components to facilitate apical constriction. These results reveal crucial links that can tie spatial information to local force generation to drive morphogenesis.

Lamellipodia are crucial for haptotactic sensing and response.

Haptotaxis is the process by which cells respond to gradients of substrate-bound cues, such as extracellular matrix proteins (ECM); however, the cellular mechanism of this response remains poorly understood and has mainly been studied by comparing cell behavior on uniform ECMs with different concentrations of components. To study haptotaxis in response to gradients, we utilized microfluidic chambers to generate gradients of the ECM protein fibronectin, and imaged the cell migration response. Lamellipodia are fan-shaped protrusions that are common in migrating cells. Here, we define a new function for lamellipodia and the cellular mechanism required for haptotaxis - differential actin and lamellipodial protrusion dynamics lead to biased cell migration. Modest differences in lamellipodial dynamics occurring over time periods of seconds to minutes are summed over hours to produce differential whole cell movement towards higher concentrations of fibronectin. We identify a specific subset of lamellipodia regulators as being crucial for haptotaxis. Numerous studies have linked components of this pathway to cancer metastasis and, consistent with this, we find that expression of the oncogenic Rac1 P29S mutation abrogates haptotaxis. Finally, we show that haptotaxis also operates through this pathway in 3D environments.

Ratiometric Imaging Using a Single Dye Enables Simultaneous Visualization of Rac1 and Cdc42 Activation.

Biosensors that report endogenous protein activity in vivo can be based on environment-sensing fluorescent dyes. The dyes can be attached to reagents that bind selectively to a specific conformation of the targeted protein, such that binding leads to a fluorescence change. Dyes that are sufficiently bright for use at low, nonperturbing intracellular concentrations typically undergo changes in intensity rather than the shifts in excitation or emission maxima that would enable precise quantitation through ratiometric imaging. We report here mero199, an environment-sensing dye that undergoes a 33 nm solvent-dependent shift in excitation. The dye was used to generate a ratiometric biosensor of Cdc42 (CRIB199) without the need for additional fluorophores. CRIB199 was used in the same cell with a FRET sensor of Rac1 activation to simultaneously observe Cdc42 and Rac1 activity in cellular protrusions, indicating that Rac1 but not Cdc42 activity was reduced during tail retraction, and specific protrusions had reduced Cdc42 activity. A novel program (EdgeProps) used to correlate localized activation with cell edge dynamics indicated that Rac1 was specifically reduced during retraction.

The regulation of RhoA at focal adhesions by StarD13 is important for astrocytoma cell motility.

Malignant astrocytomas are highly invasive into adjacent and distant regions of the normal brain. Rho GTPases are small monomeric G proteins that play important roles in cytoskeleton rearrangement, cell motility, and tumor invasion. In the present study, we show that the knock down of StarD13, a GTPase activating protein (GAP) for RhoA and Cdc42, inhibits astrocytoma cell migration through modulating focal adhesion dynamics and cell adhesion. This effect is mediated by the resulting constitutive activation of RhoA and the subsequent indirect inhibition of Rac. Using Total Internal Reflection Fluorescence (TIRF)-based Förster Resonance Energy Transfer (FRET), we show that RhoA activity localizes with focal adhesions at the basal surface of astrocytoma cells. Moreover, the knock down of StarD13 inhibits the cycling of RhoA activation at the rear edge of cells, which makes them defective in retracting their tail. This study highlights the importance of the regulation of RhoA activity in focal adhesions of astrocytoma cells and establishes StarD13 as a GAP playing a major role in this process.

SmgGDS is a guanine nucleotide exchange factor that specifically activates RhoA and RhoC.

SmgGDS is an atypical guanine nucleotide exchange factor (GEF) that promotes both cell proliferation and migration and is up-regulated in several types of cancer. SmgGDS has been previously shown to activate a wide variety of small GTPases, including the Ras family members Rap1a, Rap1b, and K-Ras, as well as the Rho family members Cdc42, Rac1, Rac2, RhoA, and RhoB. In contrast, here we show that SmgGDS exclusively activates RhoA and RhoC among a large panel of purified GTPases. Consistent with the well known properties of GEFs, this activation is catalytic, and SmgGDS preferentially binds to nucleotide-depleted RhoA relative to either GDP- or GTPγS-bound forms. However, mutational analyses indicate that SmgGDS utilizes a distinct exchange mechanism compared with canonical GEFs and in contrast to known GEFs requires RhoA to retain a polybasic region for activation. A homology model of SmgGDS highlights an electronegative surface patch and a highly conserved binding groove. Mutation of either area ablates the ability of SmgGDS to activate RhoA. Finally, the in vitro specificity of SmgGDS for RhoA and RhoC is retained in cells. Together, these results indicate that SmgGDS is a bona fide GEF that specifically activates RhoA and RhoC through a unique mechanism not used by other Rho family exchange factors.