Optogenetic Induction of Subcellular Ca2+ Events in Megakaryocytes and Platelets Using a Highly Ca2+-conductive Channelrhodopsin

doi:10.21203/rs.3.rs-3245973/v1

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Optogenetic Induction of Subcellular Ca2+ Events in Megakaryocytes and Platelets Using a Highly Ca2+-conductive Channelrhodopsin

https://doi.org/10.21203/rs.3.rs-3245973/v1

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Calcium signaling is crucial across various cell types, but its spatiotemporal dynamics remain difficult to study due to limited methods. Optogenetics, with its high precision, can address this challenge. In this study, we introduced the channelrhodopsin variant ChR2 XXM2.0, which exhibits high light sensitivity and enhanced Ca²⁺ conductance in Xenopus oocytes, into bone marrow-derived megakaryocytes through viral transduction, aiming to clarify the poorly understood role of Ca²⁺ dynamics in these cells. ChR2 XXM2.0 expression was confirmed in megakaryocyte membranes, and its functionality validated through whole-cell patch-clamp and calcium imaging. Localized activation of ChR2 XXM2.0 at the cell periphery induced cell polarization, dependent on localized calcium influx, myosin IIA, and integrin αIIbβ3-fibrinogen interaction. Furthermore, we generated a transgenic mouse line with Pf4-Cre-dependent expression of ChR2 XXM2.0, enabling optogenetic manipulation of anucleate blood platelets via light-triggered calcium signaling. Illumination induced phosphatidylserine and P-selectin exposure in spread platelets. Our results highlight the importance of asymmetric subcellular calcium events in megakaryocyte polarity and demonstrate the feasibility of manipulating platelet function using optogenetics. Taken together, our study introduces the ChR2 XXM2.0 construct and its corresponding Cre-dependent transgenic mouse line as powerful tools for manipulating subcellular Ca²⁺ signaling, with potential applications for different cell types.

Biological sciences/Cell biology/Cell polarity

Biological sciences/Physiology/Cardiovascular biology

megakaryocytes

transgenic mice

optogenetics

channelrhodopsin

cell polarization

subcellular calcium signaling

platelets

The application of optogenetics originated in neuroscience and has transformed the field over the past decades¹. Recently, it has expanded to other areas, such as plant biology and the cardiovascular system^2,3. The popular optogenetic tool, Channelrhodopsin 2 (ChR2), was discovered in the green alga Chlamydomonas reinhardtii as a light-gated cation channel mediating phototaxis behavior^4,5. It has been successfully utilized for manipulation of membrane potential in neurons at the millisecond temporal level^4,6,7 and a red-shifted Channelrhodopsin (ChR), ChrimsonR, has been clinically applied in a blind patient for partial recovery of visual function⁸. However, ChR2 is a non-selective cationic channel protein, limiting its application when ion selectivity is crucial. Calcium (Ca²⁺) plays a pivotal role in many fundamental cellular processes across virtually all cell types. To explore the spatiotemporal dynamics of Ca²⁺ signaling in cell function, a wide range of ChR variants (ChR2 H134R⁴, CatCh⁹, ChR2 XXM¹⁰, PsCatCh 2.0¹¹ and CapChR2^12,13) has been generated with improved Ca²⁺ conductance. Notably, we engineered a channelrhodopsin variant, ChR2 XXM2.0, from the ChR2 XXM¹⁰ construct, introducing a further H134Q mutation and optimizing plasma membrane-targeted expression by addition of signal peptides and N terminal truncation¹⁴. The ChR2 XXM2.0 was recently proven to be a powerful tool to study Ca²⁺ signaling in plant cells¹⁴.

In this study, we demonstrate that ChR2 XXM2.0 expressed in Xenopus oocytes exhibits high light sensitivity and significantly higher Ca²⁺-current compared to ChR2 H134R and several other Ca²⁺-permeable ChR variants, highlighting its potential as a tool to investigate spatiotemporal Ca²⁺ signaling. Increasing evidence suggests that intracellular Ca²⁺ influx in bone marrow megakaryocytes (MKs) contributes to thrombopoiesis^15–17, yet its precise spatiotemporal role in this process remains incompletely understood. We expressed ChR2 XXM2.0 in cultured bone marrow-derived MKs following viral infection and found that local Ca²⁺ influx triggers polarized MK movement in vitro, suggesting a potential mechanism underlying MK polarization at sinusoidal blood vessels in vivo.

Transgenic mice engineered to express optogenetic tools in a cell type-specific manner offer a powerful approach for exploring the roles of molecules or proteins within particular cell types, especially anucleate cells, which are otherwise challenging to target. Among the existing ChR variants, only the ChR2 H134R variant with weak Ca²⁺ conductance⁴ has been used to generate a publicly available knock-in mouse line engineered for Cre-dependent expression of ChR2 H134R¹⁸. After validating the functionality of ChR2XXM2.0 in MKs, we generated the Cre-dependent ChR2 XXM2.0 transgenic mouse line, enabling us to optogenetically target and manipulate anucleate blood platelets. Ca²⁺ plays a vital role in platelet functions, and the sustained levels of supramaximal Ca²⁺ is the prerequisite of procoagulant platelets¹⁹. The illumination of ChR2 XXM2.0-expressing platelets caused the exposure of phosphatidylserine and P-selectin, which are markers of procoagulant platelets. This work demonstrates, for the first time, the feasibility of optogenetic manipulation of anucleate blood platelets. ChR2 XXM2.0 proves to be a powerful tool for effectively regulating subcellular Ca²⁺ dynamics in MKs, platelets, and potentially in other cell types.

Comparison of different ChR2 variants for Ca²⁺ conductance in oocytes

To choose an ideal tool for Ca²⁺ modulation in primary cells, we first compared several high Ca²⁺-permeable ChR variants, especially the recently published CapChR2^12,13 and also ChR2 H134R⁴, since this ChR variant is widely used and a transgenic mouse line is available¹⁸. To compare their Ca²⁺ conductances, we measured the photocurrents in the bath solution containing 80 mM CaCl2. Before that, a final concentration of 10 mM Ca²⁺ chelator BAPTA was injected into the Xenopus oocyte to avoid the activation of Ca²⁺-activated Cl⁻ channels (CaCCs). The ChR2 H134R⁴ showed minor Ca²⁺ conductance in comparison to ChR2 XXM¹⁰, CapChR2^12,13 and ChR2 XXM2.0 (Fig. 1a, b). Notably, ChR2 XXM2.0 had the most significant Ca²⁺ current compared with ChR2 H134R⁴, XXM¹⁰ and the recently published CapChR2^12,13 (Fig. 1a, b). Instead of Ca²⁺, further recordings were also performed with Ba²⁺, which is the most similar cation to Ca²⁺ but cannot activate the endogenous CaCCs. Similarly, ChR2 XXM2.0 showed the highest current with Ba²⁺ (Fig. 1c). In comparison to ChR2 H134R⁴, all three high Ca²⁺-permeable ChR variants mutants showed prolonged off kinetics (Fig. 1d), indicating potential higher light-sensitivities¹⁰.

Functional expression of ChR2 XXM2.0 in mouse MKs

In blood platelets, the elevation of intracellular Ca²⁺ contributes to various steps of cellular activation, but its role in MK differentiation and platelet production is less well defined. In order to enable spatially and temporally controlled manipulation of Ca²⁺ signaling in MKs, we first characterized the expression and localization of ChR2 XXM2.0 in bone marrow-derived MKs after viral transduction. ChR2 XXM2.0 with an EYFP tag co-localized with glycoprotein (GP) IX, demonstrating that ChR2 XXM2.0 is expressed in the plasma membrane and internal demarcation membrane system (DMS) of MKs (Fig. 1e). To assess whether ChR2 XXM2.0 is functional in MKs, we applied whole-cell patch-clamp and measured the light-induced current in MKs. Upon illumination, a strong inward current indicating cation influx was detectable in ChR2 XXM2.0 MKs, whereas no photocurrent was detected in non-transduced MKs (Fig. 1f). We also analyzed MKs of the widely used optogenetic knock-in mouse line ChR2-EYFP with the mutation H134R¹⁸ expressing Cre recombinase under the control of the platelet factor 4 (Pf4) promoter²⁰. ChR2 H134R showed a similar expression pattern in MK membranes as compared to ChR2 XXM2.0 MKs (Supplementary Fig. 1a). However, the photocurrent in ChR2 H134R MKs was much weaker and was only ~ 8% of the ChR2 XXM2.0 MKs (Fig. 1f, Supplementary Fig. 1b). To confirm the Ca²⁺ influx, we used Cal 590™ to detect intracellular Ca²⁺ dynamics in MKs during 3 min global illumination. Increased intracellular Ca²⁺ levels were observable in ChR2 XXM2.0-positive MKs after illumination (Fig. 1g, h; Supplementary Video 1). Furthermore, spread ChR2 XXM2.0 MKs exhibited stress fiber formation and a further increase in spreading after illumination (Supplementary Fig. 2a-c). These data suggest that ChR2 XXM2.0 with enhanced Ca²⁺ permeability is functional in bone marrow-derived MKs to trigger Ca²⁺ influx.

Local activation of ChR2 XXM2.0 induces MK polarization towards the illumination side

Mature bone marrow MKs are located next to sinusoidal blood vessels and extend long cytoplasmic protrusions, designated proplatelets, into the vessel lumen, from which platelets are released. MKs need to polarize as a prerequisite for directional proplatelet release into sinusoidal vessels. Ca²⁺ has been reported to regulate cell polarity in plant cells and mammalian cells^21–23. Therefore, we performed local illumination of ChR2 XXM2.0-positive MKs spread on fibrinogen (Fig. 2a). After 3 min local illumination in a peripheral region of MKs with blue light, ChR2 XXM2.0 MKs showed the capability of directional movement comparing to control MKs as determined by the distance between the center of mass of MKs before local illumination and at the end of the observation period (Fig. 2b-d; Supplementary Video 2). The majority of ChR2 XXM2.0 MKs showed a polarization behavior towards the illumination side as evidenced by the polarization trajectories and the rose diagram (Fig. 2e, f). In contrast, ChR2 H134R MKs did not show polarized movement in response to local blue light (Supplementary Fig. 1c), suggesting a potential role of Ca²⁺ in this process. To assess the role of Ca²⁺ in light-induced polarization of ChR2 XXM2.0 MKs, spread MKs were preincubated with the cell-permeant Ca²⁺ chelator BAPTA-AM or DMSO as control before local illumination. In the presence of 100 µM BAPTA-AM, the ChR2 XXM2.0 MKs showed impaired polarization compared with the DMSO-treated group (Fig. 2g, Supplementary Video 2). Taken together, these data demonstrated that local Ca²⁺-influx is involved in polarized MK movement.

The contractile protein non-muscle myosin IIA is involved in MK polarization

Non-muscle myosin IIA is an important regulator of adhesion and polarity in cell migration. Elevation of intracellular Ca²⁺ has been shown to control cell migration through activation of myosin IIa by generating contractile forces in fish epithelial keratocytes²⁴. Furthermore, platelet migration was reported to be controlled by myosin IIA, which was activated by increased intracellular Ca^2 + 25. Thus, we hypothesized that light-induced MK polarization might also depend on non-muscle myosin IIA activity, which was supported by the increased myosin light chain 2 (MLC2) phosphorylation in ChR2 XXM2.0 MKs after illumination (Fig. 3a). To further confirm this, spread MKs were preincubated with 100 µM blebbistatin to inhibit myosin IIA function. ChR2 XXM2.0 MKs showed impaired polarization in the presence of blebbistatin compared to DMSO-treated MKs (Fig. 3b, c), suggesting that myosin IIA-dependent force generation triggered by increased intracellular Ca²⁺ is involved in light-induced ChR2 XXM2.0 MK polarization.

Local activation of ChR2 XXM2.0 triggers localized binding of integrin αIIbβ3 to fibrinogen

As a major component of platelet signaling, intracellular Ca²⁺ rise leads to platelet responses, among others to activation of the integrin αIIbβ3, which is the dominant integrin on the platelet surface²⁶. Thus, we sought to determine if the illumination of ChR2 XXM2.0 MKs results in activation of the fibrinogen receptor αIIbβ3. In order to observe integrin αIIbβ3 activation, fibrinogen labelled with Alexa Fluor 488 was added to the cell culture suspension, and a red fluorescence tag mKate2 was fused to ChR2 XXM2.0 instead of the EYFP. Subsequently, ChR2 XXM2.0-mKate2 MKs spread on fibrinogen were first globally illuminated (Fig. 4a). Illuminated MKs showed increased peripheral fibrinogen binding during the 10 min observation time, indicating activation of integrin αIIbβ3 (Fig. 4b, d). To assess the effect of polarized Ca²⁺ influx on integrin αIIbβ3 activation, ChR2 XXM2.0 expressing MKs were locally illuminated at its peripheral region (Fig. 4a). Fibrinogen binding on the plasma membrane of MKs was detectable only in the illuminated area (Fig. 4c, e), indicating polarized integrin αIIbβ3 activation. Next, we addressed whether the polarized fibrinogen binding was due to changes in the distribution of the integrin αIIbβ3, and incubated the ChR2 XXM2.0-mKate2 MKs with the anti-integrin αIIbβ3 JON6-Fab Alexa Fluor 488 antibody. However, local illumination did not change the distribution of integrin αIIbβ3 (Supplementary Fig. 3a, b), suggesting that polarized Ca²⁺ influx triggers local integrin activation rather than redistribution. To assess whether the binding between activated integrin αIIbβ3 and fibrinogen was necessary for light-induced MK polarization, spread MKs on fibrinogen were preincubated with 50 µg/ml JON/A-Fab antibody to block the activated integrin αIIbβ3 and prevent its binding to immobilized fibrinogen after local illumination. We found that blockade of the binding between integrin αIIbβ3 and fibrinogen inhibited light-induced polarized MK movement (Fig. 4f). These data demonstrated that binding of integrin αIIbβ3 to fibrinogen is important for light-induced MK polarization.

Generation of mice specifically expressing ChR2 XXM2.0 in MKs and platelets

So far, the viral approach has only allowed us to study light-induced effects on MK, not platelet function. Despite progress over the years, producing platelets under in vitro conditions remains very challenging. To establish optogenetics in platelets and manipulate platelet functions by light, we generated the optogenetic ChR2 XXM2.0 transgenic mouse line by CRISPR/Cas-mediated genome engineering. The CAG promoter-loxP-PGK-Neo-6*SV40 pA-loxP-Kozak-ChR2 XXM2.0-EYFP -rBG pA cassette was cloned into the ROSA26 locus to obtain conditional (loxP) ChR2 XXM2.0 mice, which were finally crossed with Pf4-Cre mice²⁰ to remove the stop codon and express ChR2 XXM2.0-EYFP specifically in MKs and platelets (Fig. 5a). Similar to the viral approach, ChR2 XXM2.0 was expressed in MKs of the transgenic mice (Fig. 5b). It showed high photocurrent upon global illumination (Fig. 5c) and polarized movement upon focal illumination (Fig. 5d-f, Supplementary Video 3). Successful expression of ChR2 XXM2.0 was also confirmed in platelets of transgenic mice by immunoblotting and fluorescence microscopy (Supplementary Fig. 4a, b). Phosphatidylserine (PS) exposure of platelets is induced by high sustained cytosolic Ca²⁺ flux. This prompted us to test whether platelets from homozygous ChR2 XXM2.0-EYFP^{tg/tg, Pf4−cre} mice express PS after blue light exposure. Illumination of half of the visual field resulted in a strong PS-positive signal followed by P-selectin exposure on the platelet membranes, while neighboring unilluminated platelets were unaffected (Fig. 6a-d, Supplementary Video 4). These results indicated that the ChR2 XXM2.0-EYFP platelets transition into a procoagulant state and highlighted the potential of optogenetics for manipulating the function of specific platelet subgroups within a population. Taken together, the newly generated conditional ChR2 XXM2.0 mouse line (loxP/Pf4-cre) enabled precise manipulation of cellular Ca²⁺ influx, thereby regulating MK and platelet function.

We expressed the ChR2 XXM2.0 in primary bone marrow-derived MKs and found that blue light illumination induced a significant photocurrent and Ca²⁺ signals in those cells. Local illumination of MKs triggered polarized movement on fibrinogen, which was dependent on integrin αIIbβ3-fibrinogen binding and myosin IIA activity. Further, we have established a new transgenic mouse line, which enables expression of ChR2 XXM2.0 in different cell types when combined with available Cre lines. Employing such an efficient transgenic expression strategy is especially crucial for cells like blood platelets, which would otherwise be unable to express the photosensitive protein. We capitalized on the Pf4-Cre driver line to express ChR2 XXM2.0 in platelets and found Ca²⁺-dependent phosphatidylserine and P-selectin exposure of spread platelets upon illumination. These findings demonstrate the utility of this new optogenetic transgenic mouse model for manipulating Ca²⁺ signaling and highlight its potential for broader applications.

With the help of ChR2 XXM2.0, we manipulated Ca²⁺-triggered MK polarized movement at subcellular level of a single cell for the first time. To our knowledge, this kind of manipulation has not been performed with a high Ca²⁺-conductive ChR up to now. A similar manipulation was achieved by us in plant pollen tubes which showed light-guided growth direction by activation of an anion-conducting channelrhodopsin GtACR1²⁷. Recently, several publications used different ChR2 variants to induce cell migration^12,28,29, where they utilized the temporal precision of optogenetics to generate Ca²⁺ oscillations and enhance the general motility strength at whole cell level. Our research reinforced another feature of optogenetics, the spatial precision, to dissect MK polarization at subcellular level. Optogenetic manipulation of small GTPases also achieved similar light-regulated cell polarizations, e.g. cell protrusions and ruffling³⁰. Differently, we targeted the second messenger Ca²⁺ directly and established this method in MKs. Our successful approach suggests that ChR2 XXM2.0 can be useful in other cell types to decode the physiological meaning of subcellular Ca²⁺ dynamics. Channelrhodopsins with high Ca²⁺ conductance are highly desired for the spatially and temporally precise manipulation of Ca²⁺ dynamics. Some non-channelrhodopsin-based light-gated Ca²⁺ tools show limitations of dependence on cell type-specific endogenous proteins or very slow kinetics^31–34. Recently, new Ca²⁺-permeable channelrhodopsins (CapChR) were developed with enhanced Ca²⁺ to Na⁺ conductance ratio¹². In comparison to CapChR2, ChR2 XXM2.0 showed a higher absolute Ca²⁺ current in Xenopus oocytes. The ratio of Ca²⁺ current to Na⁺ current from CapChR2 was increased to ~ 2 in the buffer containing 70 mM Ca²⁺ or 144 mM NaCl¹². However, the Na⁺ conductance can never be excluded for all the current Ca²⁺-permeable ChR variants. But the electrochemical driving force for Ca²⁺ to enter the cell is much higher than Na⁺ under the physiological conditions of mammalian cells. In our study, we also used the Ca²⁺ chelator BAPTA-AM to confirm that the light-induced effect is from the Ca²⁺ conductance of the ChR2 XXM2.0. In addition, the ChR2 H134R^4,18 with very weak Ca²⁺ conductance was able to trigger MK membrane potential depolarization, but could not evoke MK polarized movement, again showing that the high Ca²⁺ conductance is necessary for this process. Comparing to ChR2 H134R⁴ and CapChR2^12,13, ChR2 XXM2.0 showed prolonged off kinetics. Fast kinetics are highly expected in excitable cells like neurons, but in non-excitable MKs, off kinetics in the second range was fast enough for our study. More important to MKs is the application of a mild light intensity to reduce the potential photo-damage from long-time illumination.

The molecular mechanisms that orchestrate the complex process of platelet biogenesis are still incompletely understood. To identify key proteins involved in MK differentiation and platelet production, most studies so far have relied on genetically modified mouse lines or chemical reagents. To our knowledge, a reversible approach to trigger intracellular processes and directly visualize cell behavior in a high spatiotemporal resolution has not been used in MK biology until now. In this study, we used the optogenetic approach to manipulate Ca²⁺-signaling and directly observe MK behavior with high spatial resolution, and to shed new light on the role of Ca²⁺ in megakaryo- and thrombopoiesis. Our in vitro findings point towards a potential role for local Ca²⁺ rise in MK polarization towards the sinusoidal vessel, a prerequisite for platelet production. In support, it was unveiled that deletion of the glutamate-gated N-methyl-D-aspartate receptor (NMDAR) with high Ca²⁺ permeability in MKs results in reduced platelet counts in mice¹⁷, and inhibition of extracellular Ca²⁺ inflow also affected MK interaction with extracellular matrix proteins¹⁶. However, knockout mice of the major store-operated Ca²⁺ entry (SOCE) components STIM1 and Orai1 mice displayed normal platelet counts^35,36. This finding might be explained by a compensatory mechanism of different Ca²⁺ channels in MKs. We may have observed increased MK mobility in our experimental system due to the lack of neighboring cells, which typically exist in the bone marrow. In light of this, our findings rather point to a role of local elevation of intracellular Ca²⁺ in MK polarization than migration in vivo. What might cause polarized Ca²⁺ influx in MKs and lead them to polarize towards blood vessels? Studies have shown that thrombopoietin (TPO) and stromal cell-derived factor 1 (SDF-1) can induce Ca²⁺ influx in human MKs^15,37. TPO is mainly produced by the liver and transported to the bone marrow through the bloodstream, while SDF-1 is primarily generated by endothelial and perivascular mesenchymal stromal cells^38,39. The directed release of SDF-1 and TPO from the blood vessel towards the bone marrow might trigger polarized Ca²⁺ influx in MKs, causing them to polarize towards the blood vessel. We also demonstrate that the polarized Ca²⁺ influx triggers polarized MK movement in an integrin αIIbβ3-dependent manner on a fibrinogen-coated surface. Of note, fibrinogen is localized in vascular sinusoids in the bone marrow⁴⁰, and it was described that both SDF-1 and TPO increase the adhesion of megakaryoblasts to fibrinogen through activation of integrin αIIbβ3⁴¹. Thus, our findings suggest that integrin αIIbβ3 is involved in MK polarization in vivo. Interestingly, β3-integrin deficient mice display a moderately decreased platelet count^42,43. However, patients with Glanzmann thrombasthenia (inherited platelet disorder caused by mutations in the ITGA2B and ITGB3 genes encoding the αIIbβ3 integrin) present with a normal platelet count⁴⁴, but can be at the lower range of normal⁴⁵. Importantly, it has also been reported that rare gain-of-function mutations in those genes cause macrothrombocytopenia^46–51. Consistent with our data, these observations point to a potential role of αIIbβ3 in platelet biogenesis.

The conversion of activated platelets to a procoagulant state involves specific biochemical and morphological changes, including PS exposure induced by prolonged elevation of cytosolic Ca^{2+ 19,52}. ChR2 XXM2.0 precisely induced PS and P-selectin exposure on spread platelets on a fibrinogen-coated surface upon illumination. This observation demonstrates the feasibility of manipulating platelet function with optogenetic tools. We propose that the conditional (loxP) ChR2 XXM2.0 mouse line can be a useful tool to study the spatiotemporal role of Ca²⁺ influx in different cell types.

In summary, our study successfully implemented optogenetic manipulation of Ca²⁺ signaling in mouse bone marrow MKs and platelets, and revealed that local Ca²⁺ influx regulates MK polarization, pointing to a new mechanism of MK positioning towards sinusoidal vessels in vivo. The optogenetic tool ChR2 XXM2.0 and the newly developed transgenic mouse line hold promise for further investigations into subcellular Ca²⁺ signaling dynamics across diverse cell types, both in vitro and in vivo.

Mice

Mice used for experiments were at least 6 weeks old. To generate the conditional ChR2 XXM2.0-EYFP transgenic mice, the gRNA to mouse ROSA26 gene, the donor vector containing "CAG promoter-loxP-PGK-Neo-6*SV40 pA-loxP-Kozak-XXM 2.0-EYFP-rBG pA" cassette, and Cas9 mRNA were co-injected into fertilized mouse eggs to generate targeted conditional knock-in offspring by Cyagen Biosciences (Guangzhou) Inc.. F0 founder animals were identified by PCR followed by sequence analysis, which were bred to wildtype mice to test germline transmission and F1 animal generation. B6.Cg-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze/J [stock number 024109: Ai32(RCL-ChR2(H134R)/EYFP)¹⁸ transgenic mice were purchased from Jackson laboratory. Transgenic mice (with loxP sites) were intercrossed with mice carrying the Cre-recombinase under the Pf4 promoter²⁰ to generate platelet- and MK-specific knockout mice. Mice are further referred to as ChR2 XXM2.0 (+ Pf4-Cre) and ChR2 H134R (+ Pf4-Cre), respectively. Mouse bone marrow isolation was conducted according to the guidelines of the local government (Bezirksregierung Unterfranken).

DNA plasmids and viral constructs

The ChR2 H134R and XXM plasmids for Xenopus oocyte expression were from the stock of the Nagel/Gao group. The CapChR2 was generated by quick change point mutations by Chong Zhang of the Nagel/Gao group with the CoChR L112C plasmid synthesized by GeneArt Strings DNA Fragments (Life Technologies, Thermo Fisher Scientific), according to the published amino acid sequences¹³. Plasmids for Xenopus oocyte expression were linearized by NheI digestion. cRNAs were generated by in vitro transcription with the AmpliCap-MaxT7 High Yield Message Maker Kit (Epicentre Biotechnologies), using the linearized plasmid DNA as templates. The ChR2 XXM2.0 was cloned into the murine stem cell virus (MSCV) vector between the restriction sites BamHI and HindIII. The ChR2 XXM2.0-mKate2 was developed by replacing the EYFP tag of ChR2 XXM2.0 with mKate2. The EYFP was amplified by PCR with BamHI and HindIII sites on the N and C terminal, respectively, and cloned into the MSCV vector by ligation after digestion with BamHI and HindIII. Plasmids newly generated in this study will be deposited to Addgene with sequence information and will be available online when the paper is published.

Reagents and antibodies

Calcium 590™ (AAT Bioquest), DMSO (Merck), BAPTA-AM and 4’,6-Diamidino-2-Phenylindole,dihydrochloride (DAPI) (Invitrogen), blebbistatin, Fibrinogen conjugated to Alexa Fluor 488 (Sigma), anti-MLC2 p-S19 (Sigma Aldrich), anti-myosin IIA (Invitrogen), or anti-β-tubulin (Sigma Aldrich), anti-GFP and anti-GAPDH antibodies (Cell Signaling) were purchased. The antibodies JON/A (against activated form of integrin αIIbβ3, Emfret Analytics),⁵³ p0p6 (against GPIX, Emfret Analytics)⁵⁴ and JON6-Fab Alexa Fluor 488 antibody (against integrin αIIbβ3, unpublished) were modified in our laboratory.

MK culture

On day 0, bone marrow cells were obtained from femur and tibia of mice by flushing, and lineage depletion was performed using an antibody cocktail of anti-mouse CD3, clone 17A2; anti-mouse Ly-6G/Ly-6C, clone RB6-8C5; anti-mouse CD11b, clone M1/70; anti-mouse CD45R/B220, clone RA3-6B2; anti-mouse TER-119/Erythroid cells, clone Ter-119 (1.5 µg of each antibody per mouse, Biolegend) and magnetic beads (Dynabeads® Untouched Mouse CD4 Cells, Invitrogen). Lineage-negative (Lin-) cells were cultured in DMEM medium (supplemented with 10% FCS, 1% penicillin/streptomycin) containing 100 U/ml recombinant hirudin (Hyphen Biomed) and 1% TPO from supernatant (homemade) at 37°C in 5% CO₂ overnight. A bovine serum albumin density gradient was used on day 3 to separate MKs from non-MK cells. Experiments were performed on day 4.

Xenopus oocyte expression and two-electrode voltage clamp recording

cRNA-injected oocytes were incubated in ND96 solution (96 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 5 mM HEPES, pH 7.4) with 1 µM all-trans-retinal at 16°C. Two-electrode voltage-clamp (TEVC) recordings were performed 2 days after injection at room temperature.

For experiments with 80 mM Ca²⁺, 50 nl 200 mM of the fast Ca²⁺ chelator BAPTA (potassium–salt) was injected into each oocyte (~ 10 mM final concentration in the oocyte). Injected oocytes were incubated for 1 h at 16°C and then the TEVC measurement was performed. A 473 nm laser (Changchun New Industries Optoelectronics Tech) was used as light source. The light intensities were measured with a PLUS 2 Power & Energy Meter (LaserPoint s.r.l).

Expression of constructs in MKs

Targeted DNAs in the MSCV vector were transfected into 293T cells using the pCL vector system and ROTIFect from Roth. Viral supernatant was collected and bone marrow-derived cells were infected on day 1.

Immunofluorescence of MKs

To visualize protein expression and localization, cultured MKs were fixed and permeabilized with 4% PFA and 0.1% Triton-X-100 in PHEM-Buffer. The plasma membrane and demarcation membrane system were stained using Alexa 647-conjugated anti-GPIX antibody, and the nucleus with DAPI. Samples were visualized with a Leica TCS SP8 confocal microscope (Leica Microsystems).

Whole-cell patch-clamp

Whole-cell currents were recorded from a single MK using a GeneClamp-500B amplifier (Axon Instruments) with an inverted microscope (Leica DMi8). Patch pipettes were prepared from borosilicate tubes (Kimble Chase) using a PC-10 vertical puller (Narishige) and polished with an MF2 microforge (Narishige). Experiments were performed at room temperature (~ 22°C). The bath solution contained 145 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM D-glucose, 10 mM HEPES, pH 7.35. The pipette solution contained 150 mM KCl, 0.1 mM EGTA, 1 mM MgCl₂, 0.05 mM Na₂GTP, 10 mM HEPES, pH 7.2. A 473 nm laser (Changchun New Industries Optoelectronics Tech) was used as a light source. Stimulation and data acquisition were controlled with NI USB-6221 (National instruments) and WinWCP software (v4.1.7, 103 Strathclyde University, UK). Origin 2020 Pro was used for data analysis.

MK spreading

Chamber slides (µ-Slide 8 Well, uncoated, Ibidi) were coated with 100 µg/ml fibrinogen at 4°C overnight. After washing, chamber slides were blocked with 3% BSA/PBS for 1h. Subsequently, MKs were seeded in chamber slides for 3h at 37°C and 5% CO₂. The chamber slides were washed and 200 µl DMEM medium supplemented with 10% FCS, 1% penicillin/streptomycin was added.

Illumination of MKs

Spread MKs were illuminated with the 488 nm laser line (10% laser power) of the Argon laser (22% laser power) using the FRAP module of a confocal laser scanning microscope (TCS SP8 confocal, Leica Microsystems) equipped with an HCPLAPO CS2 63×/1.40 OIL objective. Region of interest covering peripheral parts of the MK (local illumination) or the entire MK (global illumination) were selected for blue light illumination to activate the ChR2 XXM2.0. HeNe 633 nm laser light (1% laser power) was used to observe MK behavior in brightfield. An area of 512 × 512 pixels was selected as the imaging region. The imaging interval was 30 s. Videos and images of MKs were generated with FIJI/IMAGEJ. MKs in suspension were illuminated with 100 µW/60 mm² LED light.

Immunoblotting

Cell lysates were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes. Blots were probed for MLC2 p-S19 (Sigma Aldrich), myosin IIA (Invitrogen), or β-tubulin (Sigma Aldrich). Horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence solution (MoBiTec) were used for visualization. Immunoblots were recorded directly using an Amersham Imager 600 (GE Healthcare).

MK polarization

The MK outline was manually drawn and the center of mass was calculated by FIJI/ImageJ. The polarization distance was calculated by the distance between the two centers of mass of MKs before local illumination and 20 min observation after illumination. Color-coded time sequence of MK outlines was manually drawn by FIJI/ImageJ. MK polarization trajectory and the rose diagram were produced by Chemotaxis and Migration Tool 2.0.

Platelet spreading and PS exposure

Slides were coated with human fibrinogen (100 µg/mL, Sigma), incubated at 4°C overnight, and blocked with 1% BSA diluted in PBS for at least 30 min at room temperature. Subsequently, slides were washed with Tyrode´s buffer with 2 mM Ca²⁺. 150,000 platelets per µL in Tyrode´s buffer with 2 mM Ca²⁺, thrombin (f.c. 0.01 U/mL), 0.35 µg/ml Annexin V Alexa Fluor 546, and 18 ug/ml anti-P-selectin Alexa Fluor 647 antibody. The platelets were spread in ibidi dish for 30 minutes before 10 min illumination.

Data analysis

Results are mean ± s.d., individual data points were graphed in the figure. The statistical significance in the whole-cell patch clamp experiments were analyzed using the student’s T-test. Differences between two groups were analyzed using the Mann–Whitney two-tailed test. For more than two groups, Kruskal–Wallis test followed by Dunn’s test for multiple comparisons was performed using GraphPad Prism software. P-values < 0.05 were considered as statistically significant: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Results with a P-value > 0.05 were considered as not significant (ns in the figures).

Methods were approved by the district government of Lower Franconia (Bezirksregierung Unterfranken) and performed in accordance with the relevant guidelines and regulations.

Data Availability Statement

The data generated and analyzed in this study are available from the corresponding authors on reasonable request.

Declaration of Interest

The authors declare that they have no conflict of interest.

Author Contributions

Performed experiments: YZ, JYS, SG; Data analysis: all authors; the ChR2 XXM2.0 transgene mouse was designed and ordered by CX and CS; Writing – original draft: YZ, SG, MB; Supervised research: GN, SG, MB; Funding acquisition: MB, GN, SG; All authors have critically revised and approved the final version of the manuscript.

Acknowledgments

We thank Bernhard Nieswandt and the microscopy platform of the Core Unit Imaging, Rudolf Virchow Center for Integrative and Translational Imaging for providing technical infrastructure and support, Shang Yang and Chong Zhang for the cloning of some Channelrhodopsin variants, and Zoltan Nagy for providing antibodies. This work was supported by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation): Project numbers TR240 374031971 to M.B and G.N., 452622672 to M.B. and 525167920 to M.B. and S.G.

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Optogenetic Induction of Subcellular Ca2+ Events in Megakaryocytes and Platelets Using a Highly Ca2+-conductive Channelrhodopsin

Status:

Version 1

Abstract

Figures

Introduction

Results

Comparison of different ChR2 variants for Ca²⁺ conductance in oocytes

Functional expression of ChR2 XXM2.0 in mouse MKs

Local activation of ChR2 XXM2.0 induces MK polarization towards the illumination side

The contractile protein non-muscle myosin IIA is involved in MK polarization

Local activation of ChR2 XXM2.0 triggers localized binding of integrin αIIbβ3 to fibrinogen

Generation of mice specifically expressing ChR2 XXM2.0 in MKs and platelets

Discussion

Materials and Methods

Mice

DNA plasmids and viral constructs

Reagents and antibodies

MK culture

Expression of constructs in MKs

Immunofluorescence of MKs

Whole-cell patch-clamp

MK spreading

Illumination of MKs

Immunoblotting

MK polarization

Platelet spreading and PS exposure

Data analysis

Declarations

Data Availability Statement

Declaration of Interest

Author Contributions

Acknowledgments

References

Additional Declarations

Supplementary Files

Status:

Version 1

Optogenetic Induction of Subcellular Ca2+ Events in Megakaryocytes and Platelets Using a Highly Ca2+-conductive Channelrhodopsin

Status:

Version 1

Abstract

Figures

Introduction

Results

Comparison of different ChR2 variants for Ca2+ conductance in oocytes

Functional expression of ChR2 XXM2.0 in mouse MKs

Local activation of ChR2 XXM2.0 induces MK polarization towards the illumination side

The contractile protein non-muscle myosin IIA is involved in MK polarization

Local activation of ChR2 XXM2.0 triggers localized binding of integrin αIIbβ3 to fibrinogen

Generation of mice specifically expressing ChR2 XXM2.0 in MKs and platelets

Discussion

Materials and Methods

Mice

DNA plasmids and viral constructs

Reagents and antibodies

MK culture

Expression of constructs in MKs

Immunofluorescence of MKs

Whole-cell patch-clamp

MK spreading

Illumination of MKs

Immunoblotting

MK polarization

Platelet spreading and PS exposure

Data analysis

Declarations

Data Availability Statement

Declaration of Interest

Author Contributions

Acknowledgments

References

Additional Declarations

Supplementary Files

Status:

Version 1

Comparison of different ChR2 variants for Ca²⁺ conductance in oocytes