Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

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This manuscript provides convincing evidence for the involvement of membrane actin, and its regulatory proteins, mDia1/3, RhoA, and Rac1 in the mechanism of synaptic vesicle re-uptake (endocytosis). These important data fill a gap in the understanding of how the regulation of actin dynamics and endocytosis are linked. The manuscript will be of interest to all scientists working on cellular trafficking and membrane remodeling.

https://doi.org/10.7554/eLife.92755.3.sa0

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Abstract
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Abstract

Neurotransmission at synapses is mediated by the fusion and subsequent endocytosis of synaptic vesicle membranes. Actin has been suggested to be required for presynaptic endocytosis but the mechanisms that control actin polymerization and its mode of action within presynaptic nerve terminals remain poorly understood. We combine optical recordings of presynaptic membrane dynamics and ultrastructural analysis with genetic and pharmacological manipulations to demonstrate that presynaptic endocytosis is controlled by actin regulatory diaphanous-related formins mDia1/3 and Rho family GTPase signaling in mouse hippocampal neurons. We show that impaired presynaptic actin assembly in the near absence of mDia1/3 and reduced RhoA activity is partly compensated by hyperactivation of Rac1. Inhibition of Rac1 signaling further aggravates impaired presynaptic endocytosis elicited by loss of mDia1/3. Our data suggest that interdependent mDia1/3-Rho and Rac1 signaling pathways cooperatively act to facilitate synaptic vesicle endocytosis by controlling presynaptic F-actin.

Introduction

Synaptic transmission relies on the release of neurotransmitters by calcium-triggered exocytic fusion of synaptic vesicles (SVs) at specialized active zone release sites within presynaptic nerve terminals. Following fusion, compensatory endocytosis retrieves SV proteins and lipids from the presynaptic plasma membrane and SVs are reformed (Chanaday et al., 2019; Gan and Watanabe, 2018; Kononenko and Haucke, 2015; Saheki and De Camilli, 2012; Soykan et al., 2016), e.g., by clathrin-mediated vesicle budding (Kononenko et al., 2014; Watanabe et al., 2014) (but see Wu et al., 2014 for evidence in favor of clathrin-independent SV reformation). Endocytosis relies on mechanical forces to enable membrane deformation and eventually fission (Engqvist-Goldstein and Drubin, 2003). In many biological systems, actin and actin-associated myosin motors appear to provide such mechanical force to facilitate vesicle formation (Anes et al., 2003; Marston et al., 2003). For example, the function of actin for endocytosis is well-established in yeast, whereas differential requirements for actin in distinct forms of endocytosis have been described in various types of mammalian cells including neurons (Boulant et al., 2011; Merrifield et al., 2005; Saffarian et al., 2009; Saheki and De Camilli, 2012). Pharmacological inhibition of actin assembly by latrunculin has been shown to cause the accumulation of endocytic intermediates at lamprey giant synapses (Shupliakov et al., 2002) and interfere with ultrafast endocytosis in response to single optical action potentials (APs) at hippocampal synapses (Watanabe et al., 2013; Watanabe et al., 2014; Wu and Chan, 2022) and with fast endocytosis at cerebellar mossy fiber boutons (Delvendahl et al., 2016). The same treatment does not hamper endocytosis of SV proteins at hippocampal synapses stimulated with trains of APs (Sankaranarayanan et al., 2003; Soykan et al., 2017). Interference with actin function by conditional knockout (KO) of Actb or Actg1 genes, encoding β- or γ-actin, respectively, has suggested a crucial role for actin in all kinetically distinguishable forms of endocytosis (Wu et al., 2016) at hippocampal synapses and the calyx of Held, a fast giant synapse in the auditory brain stem (Borst and Soria van Hoeve, 2012). However, in the same preparations, actin loss also affected SV exocytosis (Wu et al., 2016), suggesting a more general requirement of actin for presynaptic function. Consistently, actin has been shown to surround clusters of reserve pool SVs in lamprey (Bloom et al., 2003), to facilitate the replenishment of fast-releasing vesicles in various models (Sakaba et al., 2013; Sakaba and Neher, 2003), to be involved in Tau pathology (Zhou et al., 2017), to induce spine growth and plasticity, (Cingolani and Goda, 2008) and to steer neuronal migration (Shinohara et al., 2012) among various other roles.

We have previously demonstrated that pharmacological interference with the function of formins, a group of Rho family-associated proteins that nucleate linear filamentous (F) actin, kinetically delays SV endocytosis at hippocampal boutons and blocks compensatory endocytosis at the calyx of Held in pre-hearing rats (Soykan et al., 2017). Capacitance measurements at the calyx of Held in post-hearing rodents suggest a less stringent requirement for formin-mediated actin assembly for endocytosis (Hori et al., 2022). The latter observation may reflect the operation of compensatory pathways for actin assembly, e.g., via Rac1 or Cdc42 Rho-family GTPases that promote branched actin networks (Eisenmann et al., 2005; Goode and Eck, 2007; Hodge and Ridley, 2016).

In contrast to the vast body of literature regarding the role of actin and actin-associated proteins at the postsynapse (Cingolani and Goda, 2008; Colgan and Yasuda, 2014) and references therein, most notably the actin-rich mesh of the postsynaptic density of glutamatergic synapses, comparably little is known about the signaling pathways e.g., via guanine nucleotide exchange factors and GTPase activating proteins for Rho family small GTPases (Müller et al., 2020) that mediate actin assembly at the presynapse. Recent work has suggested that presynaptic Rac1 negatively regulates synaptic strength and release probability by altering SV priming and replenishment at central synapses (O’Neil et al., 2021) including the calyx of Held (Keine et al., 2022). These data suggest a negative regulatory role for Rac1 in SV exocytosis. The abundance of postsynaptic actin (Chen et al., 2020; Cingolani and Goda, 2008; Colgan and Yasuda, 2014) has hampered the analysis of the nanoscale localization of actin at presynaptic nerve terminals and of the actin-regulatory proteins that control its dynamics. Loss of dynamin isoforms Dynamin1 and Dynamin3, the main enzymes for endocytic membrane fission (Ferguson et al., 2007; Imoto et al., 2022; Raimondi et al., 2011), has been shown to cause the accumulation of F-actin at and around stalled endocytic intermediates in non-neuronal cells (Ferguson et al., 2009) but this phenotype seems less overt at hippocampal synapses (Raimondi et al., 2011). Hence, actin in addition to a possible local role at endocytic invaginations might serve a more general function in endocytosis at synapses, for example by modulating plasma membrane tension or contractility, by providing a restrictive membrane scaffold that promotes membrane fission by keeping endocytic invaginations under longitudinal tension (Boulant et al., 2011; Roux et al., 2006; Wu and Chan, 2022), or via formation of an F-actin ring around the active zone that mechanically couples exocytic membrane compression to endocytic pit formation (Ogunmowo et al., 2023).

Here, we combine optical recordings of SV exo-/endocytosis and ultrastructural analysis with genetic and pharmacological manipulations to show that interdependent mDia1/3-Rho and Rac1 signaling pathways cooperate to facilitate SV endocytosis by controlling presynaptic F-actin.

Results

Actin dynamics and actin nucleating mDia1/3 proteins facilitate presynaptic endocytosis and SV recycling

Previous studies using pharmacological inhibitors of actin polymerization and depolymerization have yielded inconclusive, often conflicting data regarding the function of actin in SV endocytosis and recycling in different models (Bleckert et al., 2012; Del Signore et al., 2021; Piriya Ananda Babu et al., 2020; Sankaranarayanan et al., 2003; Shupliakov et al., 2002; Wu and Chan, 2022). In contrast, ablation of Actb or Actg1in mouse neurons suggests that actin is required for all forms of endocytosis at several types of synapses (Wu et al., 2016). Prompted by these findings we revisited the role of actin in presynaptic endocytosis by analyzing the effects of impaired F-actin dynamics in the combined presence of the G-actin sequestering drug latrunculin A, the F-actin stabilizer jasplakinolide, and Y-27632, an inhibitor of ROCK kinase signaling. This cocktail was shown to preserve the existing cytoskeleton architecture while blocking actin assembly, disassembly, and rearrangement (Peng et al., 2011). We optically recorded the stimulation-induced exo-endocytosis of the SV protein Synaptophysin (Syph) fused to a pH-sensitive super-ecliptic green fluorescent protein (pHluorin) (Miesenböck et al., 1998) that is de-quenched during exocytosis and undergoes re-quenching as SVs are internalized and re-acidified in hippocampal neurons at physiological temperature. Under these conditions (i.e. trains of APs, 37 °C), SV endocytosis occurs on a timescale of >10 s, e.g., a timescale that is slower than re-quenching of pHluorin due to reacidification (López-Hernández et al., 2022; Soykan et al., 2017). Hence, the decay of pHluorin signals can serve as a measure of the time course of SV endocytosis. Perturbation of actin dynamics in the combined presence of latrunculin A, jasplakinolide, and Y-27632 significantly slowed down the endocytic retrieval of exogenously expressed Syph-pHluorin (Figure 1A and B). Application of Y-27632 alone or a combination of latrunculin A and jasplakinolide had no effect on SV endocytosis kinetics, whereas combined use of Y-27632 together with jasplakinolide displayed a mild inhibitory effect (Figure 1—figure supplement 1C–F). Impaired actin dynamics in the presence of latrunculin A, jasplakinolide, and Y-27632 did not impact the apparent level of SV exocytosis indicated by the maximal amplitude of Syph-pHluorin fluorescence (Figure 1—figure supplement 1A). As optical imaging of pHluorin reporters may lead to artifacts, we analyzed the internalization kinetics of the endogenous vesicular γ-aminobutyric acid transporter (vGAT) using antibodies directed against its luminal domain coupled to the pH-sensitive fluorophore CypHer5E (Hua et al., 2011; López-Hernández et al., 2022). The cyanine-based dye CypHer5E is quenched at neutral pH but exhibits bright fluorescence when present in the acidic lumen of SVs and, thus can serve as a tracer for the exo-endocytic cycling of endogenous SV proteins. Perturbation of actin dynamics significantly delayed the endocytic retrieval of endogenous vGAT in response to train stimulation with 200 APs at physiological temperature (Figure 1C and D), consistent with the results from exogenously expressed Syph-pHluorin. vGAT exocytosis proceeded unperturbed (Figure 1—figure supplement 1B). Hence, a dynamic actin cytoskeleton facilitates presynaptic endocytosis in hippocampal neurons.

Figure 1 with 1 supplement see all

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Actin dynamics and actin-nucleating mDia1/3 proteins facilitate synaptic vesicle (SV) endocytosis.

(A) Averaged normalized Synaptophysin-pHluorin (Syph-pH) fluorescence traces from transfected hippocampal neurons stimulated with 200 action potentials (APs) (40 Hz, 5 s) at physiological temperature (37.5 °C). Neurons were treated with 0.1% dimethyl sulfoxide (DMSO) or JLY cocktail (containing 8 µM Jasplakinolide, 5 µM Latrunculin A, and 10 µM Y-27632) as indicated. Data shown represent the mean ± SEM. N=4 independent experiments from n_DMSO = 23 videos; n_JLY = 36 videos. (B) Endocytic decay constants (τ) of Synaptophysin-pHluorin traces in A: τ_DMSO = 29.1±3.4 s; τ_JLY = 55.8±7.2 s; p<0.05, two-tailed student’s t-test. Data shown represent mean ± SEM. (C) Averaged normalized bleach-corrected vGAT-CypHer fluorescence traces from hippocampal neurons treated with DMSO or JLY cocktail in response to 200 AP (40 Hz, 5 s) stimulation. Data shown represent the mean ± SEM. N=4 independent experiments from n_DMSO = 23 videos; n_JLY = 29 videos. (D) Endocytic decay constants of vGAT-CypHer traces in C: τ_DMSO = 10.9±0.7 s, τ_JLY = 24.6±2.0 s; p<0.001, two-tailed unpaired student’s t-test. Data shown represent the mean ± SEM. (E) Averaged normalized Synaptophysin-pHluorin fluorescence traces from hippocampal neurons transfected with shRNA-encoding plasmids against no mammalian target (*shCTR*) or *Diaph1* (*shmDia1*) in response to 200 AP (40 Hz, 5 s) stimulation. Neurons were co-transfected with mDia1-mCherry (mDia1-WT) or mCherry alone (*shCTR* & *shmDia1*) to exclude artifacts from overexpression. Data shown represent the mean ± SEM. N=3 independent experiments from n_shCTR = 28 videos, n_shmDia1=21 videos, n_{shmDia1 + mDia1-WT}=21 videos. (F) Endocytic decay constants of Synaptophysin-pHluorin traces in E: τ_shCTR = 29.7±1.9 s; τ_shmDia1 = 64.7 ± 3.9 s; τ_{shmDia1 + mDia1-WT} = 30.6±3.7 s; p_{shCTR vs shmDia1} <0.001, p_{shmDia1 vs shmDia1 + mDia1-WT}<0.001, one-way ANOVA with Tukey’s post-test. Data shown represent mean ± SEM. (G) Endocytic decay constants of averaged normalized vesicular glutamate transporter 1 (vGLUT1)-pHluorin fluorescence traces (Figure 1—figure supplement 1I) from hippocampal neurons transduced with *shCTR* (τ_shCTR = 9.1±0.8 s), *shmDia1* (τ_shmDia1 = 14.3±1.5 s), or *shmDia1 +3* (τ_shmDia1+3 = 16.4±1.3 s) in response to 40 AP (20 Hz, 2 s) stimulation (p_{shCTR vs shmDia1} <0.05, p_{shCTR vs shmDia1+3} < 0.01, one-way ANOVA with Tukey’s post-test). Data shown represent mean ± SEM. N=4 independent experiments from n_shCTR = 17 videos; n_shmDia1=19 videos; n_shmDia1+3 = 18 videos. (H) Endocytic decay constants of averaged normalized vGLUT1-pHluorin fluorescence traces (Figure 1—figure supplement 1K) of neurons transduced with lentiviral vectors encoding *shCTR* (τ_shCTR = 13.6±1.6 s), *shmDia1* (τ_shmDia1 = 22.0±3.2 s) or *shmDia1 +3* (τ_shmDia1+3 = 26.9±3.6 s) in response to 80 AP (40 Hz, 2 s) stimulation (p_{shCTR vs shmDia1+3} < 0.05, one-way ANOVA with Tukey’s post-test). Data shown represent mean ± SEM. N=4 independent experiments from n_shCTR = 12 videos, n_shmDia1=15 videos, n_shmDia1+3 = 18 videos. (I) Averaged normalized bleach-corrected vGAT-CypHer fluorescence traces from hippocampal neurons transduced with *shCTR* or *shmDia1 +3* in response to 200 AP (40 Hz, 5 s) stimulation. Data shown represent the mean ± SEM. N=8 independent experiments from n_shCTR = 37 videos, n_shmDia1+3 = 35 videos. (J) Endocytic decay constants of vesicular γ aminobutyric acid transporter (vGAT)-CypHer traces in I: τ_shCTR = 14.1±1.3 s; τ_shmDia1+3 = 27.3±2.6 s; p<0.001, two-tailed unpaired student’s t-test. Data shown represent the mean ± SEM. (K) Averaged normalized vGLUT1-pHluorin fluorescence traces from transduced neurons in response to 80 AP (40 Hz, 2 s) stimulation. Cells were treated with 0.1% DMSO or 10 µM mDia activator (IMM) in the imaging buffer. Data shown represent mean ± SEM. N=3 independent experiments from n_DMSO = 18 videos; n_IMM = 16 videos. (L) Endocytic decay constants of vGLUT1-pHluorin traces in K: τ_DMSO = 14.9±0.8 s; τ_IMM = 9.8±0.5 s; p<0.05, two-tailed unpaired student’s t-test. Data shown represent mean ± SEM.

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Previous studies have suggested that formins, in particular mDia1, promote SV endocytosis (Soykan et al., 2017), likely by inducing F-actin assembly (Ganguly et al., 2015). We confirmed that shRNA-mediated depletion of Diaph1, encoding mDia1 (shmDia1), from hippocampal neurons led to significantly delayed Syph-pHluorin endocytosis and re-acidification in response to stimulation with 200 APs. Re-expression of shRNA-resistant wild-type mDia1 fully restored normal endocytosis kinetics (Figure 1E and F). Impaired SV endocytosis in lentivirus-mediated mDia1-depleted neurons (Figure 1—figure supplement 1H) was also observed, if SV exo-/endocytosis was triggered by stimulation with 40 APs or 80 APs and probed with vesicular glutamate transporter 1 (vGLUT1)-pHluorin (Figure 1G and H and Figure 1—figure supplement 1I and K). Exocytic fusion of vGLUT1-pHluorin-containing SVs was unaffected (Figure 1—figure supplement 1G). Co-depletion of the closely related mDia3 isoform tended to further aggravate this phenotype (Figure 1G and H and Figure 1—figure supplement 1I and K) without impacting the apparent number of fused SVs (Figure 1—figure supplement 1J and L) or vGAT exocytosis (Figure 1—figure supplement 1M). Furthermore, mDia1/3 depletion significantly delayed the endocytic retrieval of endogenous vGAT (Figure 1I and J). Conversely, the application of IMM-01, a small molecule activator of mDia-related formins that acts by relieving autoinhibition (Lash et al., 2013; Figure 1—figure supplement 1N), led to a moderate but significant acceleration of SV endocytosis monitored by vGLUT1-pHluorin (Figure 1K and L) but no change in apparent SV fusion (Figure 1—figure supplement 1O). Ultrastructural analysis of hippocampal synapses from mDia1/3-depleted neurons by electron microscopy revealed a reduction in SV density compared to controls (Figure 2A and B). This phenotype was rescued upon prior silencing of endogenous neuronal network activity in the presence of tetrodotoxin (TTX) (Figure 2C and Figure 2—figure supplement 1A and B). A similar, slightly more pronounced depletion of SVs was observed in hippocampal synapses from Diaph1 knockout (mDia1KO) mice (Peng et al., 2007; Peng et al., 2003; Shinohara et al., 2012; Figure 2D and E).

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Loss of mDia1/3 causes an activity-dependent reduction of the synaptic vesicle (SV) pool.

(A) Representative synaptic electron micrographs from hippocampal neurons transduced with lentiviruses encoding *shCTR* or *shmDia1 +3*, targeting *Diaph1/2* genes. Postsynapse and postsynaptic cleft are colored in green and maroon, respectively. Scale bar, 250 nm. (B) Average number of SVs per μm² in boutons from *shCTR* (92.2±2.5) and *shmDia1 +3* (81.4±2.9; p<0.0001, Mann-Whitney test) treated neurons. Data shown represent mean ± SEM. N=3 independent experiments from n_shCTR = 326 synapses, n_shmDia1+3 = 321 synapses. (C) Average number of SVs per μm² in synaptic boutons from hippocampal neurons transduced with lentiviruses encoding *shmDia1 +3* and treated with 0.1% Vehicle (10 µM NaOAc; 83.2±2.9) or 1 µM Tetrodotoxin (TTX; 93.8±3.1; p<0.01, Mann-Whitney test) for 36 hr before chemical fixation. Data shown represent mean ± SEM from two independent experiments and n_Vehicle = 225 synapses, n_TTX = 221 synapses. Representative synaptic electron micrographs are shown in Figure 2—figure supplement 1A. The dotted line represents the average SV numbers/μm² in *shCTR* boutons treated with dimethyl sulfoxide (DMSO) from the same experiments as a reference (Figure 2—figure supplement 1A), (B). (D) Representative electron micrographs of synapses in hippocampal neurons from wild-type (WT) or *Diaph1* (encoding mDia1) knockout (KO) mice. Postsynapse and postsynaptic cleft are colored in green and maroon, respectively. Scale bar, 250 nm. (E) Average number of SVs per μm² in WT (117.6±5.3) and *mDia1* KO (84.6±5.2; p<0.0001, Mann-Whitney test) boutons. Data shown represent the mean ± SEM from n_WT = 103, n_mDia1KO = 96 synapses (N=1).

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These data are consistent with a role for actin dynamics (see Figure 1) and actin nucleating mDia1/3 proteins in presynaptic endocytosis and SV recycling.

mDia facilitates SV endocytosis by regulating presynaptic F-actin

mDia1 in addition to its Rho binding domain and a C-terminal actin-assembling formin homology 2 (FH2) domain comprises an unstructured N-terminal region involved in membrane binding (Eisenmann et al., 2005; Ramalingam et al., 2010; Figure 3A). This architecture suggests that mDia specifically promotes the nucleation of unbranched actin filaments at membranes, for example, to promote endocytosis at the presynaptic plasma membrane. To test this hypothesis, we assayed the ability of mutant mDia1 lacking its N-terminal membrane-binding region (mDia1-ΔN) (Ramalingam et al., 2010) to rescue defective SV endocytosis in hippocampal neurons depleted of endogenous mDia1. Mutant mDia1-ΔN displayed reduced association with membranes in transfected HEK293T cells (Figure 3—figure supplement 1A and B) and was unable to restore normal kinetics of Syph-pHluorin endocytosis in hippocampal neurons transfected with Diaph1-specific microRNA (shmDia1miR; Figure 3B and Figure 3—figure supplement 1C). Hence, mDia may act on SV endocytosis by specifically promoting actin assembly at presynaptic membranes. We probed this model at several levels. We analyzed the nanoscale localization and distribution of mDia1 via multicolor time-gated stimulated emission depletion (gSTED) microscopy. Hippocampal neurons were stained for mDia1, the presynaptic active zone marker Bassoon, and post-synaptic Homer1. The distributions of all three proteins along line profiles perpendicular to the synaptic cleft (i.e. the space between Bassoon and Homer1 clusters) were analyzed (Gerth et al., 2017; Figure 3—figure supplement 1H). This analysis revealed that endogenous mDia1 is primarily localized to the presynaptic compartment, where it is concentrated at or very close to the plasma membrane (Figure 3C and D), i.e., <50 nm away from the presynaptic membrane see Dani et al., 2010. Prompted by these results we dissected the interactome of mDia1 by unbiased quantitative proteomic analyses of immunoprecipiates from detergent-extracted mouse synaptosomes (Figure 3—figure supplement 1D). In addition to actin, we identified a variety of additional actin-regulatory proteins including Myosin IIB (Chandrasekar et al., 2013) and the small GTPase Rac1 (Figure 3E). We confirmed the association of mDia1 with actin and Myosin IIB (Figure 3—figure supplement 1E) and found Myosin IIB to be concentrated at or near the presynaptic plasma membrane (Figure 3—figure supplement 1F and G) akin to the localization of mDia1. The association of mDia1 with Myosin IIB is consistent with the inhibitory effects of the Myosin II inhibitor blebbistatin on SV endocytosis (Chandrasekar et al., 2013; Soykan et al., 2017). Interestingly, we also observed an enrichment of several endocytic proteins previously implicated in SV recycling in mDia1 immunoprecipitates. These include the lipid phosphatase Synaptojanin1, the bin-amphiphysin-rvs (BAR) domain proteins Amphiphysin, Endophilin A1, and PACSIN1/2 and, the GTPase Dynamin1 (Chanaday et al., 2019; Gan and Watanabe, 2018; Saheki and De Camilli, 2012; Soykan et al., 2016; Figure 3E) as confirmed by immunoblotting (Figure 3F). Based on the association of mDia1 with endocytic proteins including Dynamin1, we hypothesized that mDia1 might get stalled at endocytic intermediates that accumulate under conditions of perturbed Dynamin function (Ferguson et al., 2009; Raimondi et al., 2011). Application of the partially non-selective Dynamin inhibitor Dynasore (Macia et al., 2006; Figure 3C and H) or expression of dominant-negative mutant Dynamin1 K44A (Figure 3G1) led to a significant accumulation of mDia1 at presynaptic sites marked by Bassoon. mDia1 levels at the postsynapse were also increased (Figure 3—figure supplement 1I). Dynasore did not impact the levels (Figure 3—figure supplement 1J) or distribution of Bassoon or Homer1.

Figure 3 with 1 supplement see all

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mDia1 associates with endocytic proteins and localizes to presynaptic sites.

(A) Schematic representation of functional domains of mDia1. Rho-binding domain (RBD), Diaphanous inhibitory domain (DID), Dimerization domain (DD), Coiled coil domain (CC), Formin homology domain 1 (FH1), Formin homology domain 2 (FH2), Diaphanous autoinhibitory domain (DAD). The unstructured N-terminus (first 60 amino acids) contains three basic stretches and was truncated in the ΔN mutant. (B) Endocytic decay constants of Synaptophysin-pHluorin traces (Figure 3—figure supplement 1C) from hippocampal neurons transfected with shRNAmiR against no mammalian target (*shCTRmiR*) or *Diaph1* (*shmDia1miR*) in response to 200 action potential (AP) (40 Hz, 5 s) stimulation. For rescue experiments, neurons were co-transfected with plasmids encoding mDia1-WT-mCherry (τ_{shmDia1miR + mDia1-WT}=20.0±0.8 s), mDia1-ΔN-mCherry (τ_{shmDia1miR + mDia1-ΔN}=34.5±2.9 s), or mCherry alone (τ_shCTRmiR = 21.8±1.1 s, τ_shmDia1miR = 30.4±1.9 s) to exclude artifacts from overexpression (p_{shCTRmiR vs shmDia1miR} < 0.05; p_{shmDia1miR vs shmDia1miR + mDia1-WT}<0.01; p_{shmDia1miR + mDia1-WT vs shmDia1miR + mDia1-ΔN}<0.01, one-way ANOVA with Tukey’s post-test). Data shown represent mean ± SEM. N=5 independent experiments from n_shCTRmiR = 41 videos, n_shmDia1miR = 51 videos, n_{shmDia1miR + mDia1-WT}=35 videos, n_{shmDia1miR + mDia1-ΔN}=37 videos. (C) Representative three-channel time-gated stimulated emission depletion (STED) images of synapses from hippocampal cultures treated with 0.1% dimethyl sulfoxide (DMSO) or 80 μM Dynasore for 10 min before fixation and immunostained for Bassoon (presynaptic marker, magenta), mDia1 (cyan) and Homer1 (postsynaptic marker, green). Scale bar, 250 nm. (D) Averaged normalized line profiles for synaptic distribution of mDia1 and Homer1 relative to Bassoon (Maximum set to 0 nm). Data represent mean ± SEM. N=3 independent experiments from n=235 synapses. (E) Volcano plot of proteins associating with synaptic mDia1 analyzed by label-free proteomics of anti-mDia1 versus control (CTR) immunoprecipitates from detergent-extracted mouse synaptosomes (P2’ fraction). The logarithmic ratios of protein intensities are plotted against negative logarithmic p-values derived from a two-tailed student’s t-test. N=3 independent experiments. Each dot represents one protein. Selected cytoskeletal hits include: Actin, Myosin IIB (MyoIIB), and Rac1. Selected endocytic hits include Amphiphysin (p<0.05), Dynamin1, Endophilin-A1, PACSIN1, PACSIN2 (p<0.05), and Synaptojanin1. (F) Endogenous immunoprecipitation of mDia1 from detergent-extracted mouse synaptosomes (P2’ fraction) using mDia1-specific antibodies. Immunoprecipitates were analyzed by immunoblotting for mDia1, Dynamin1, and β-Actin. (G) Representative three-channel time-gated STED images of synapses from hippocampal cultures transduced with wild-type Dynamin1 (WT) or GTPase-deficient Dynamin1 (K44A) in response to 200 AP (40 Hz, 5 s) stimulation. Cells were immunostained for Bassoon (magenta), mDia1 (cyan), and Homer1 (green). Scale bar, 250 nm. (H) Presynaptic mDia1 levels in synapses treated with 0.1% DMSO (100±7.3) or 80 µM Dynasore (145.8±9.3; p=0.0001; one sample Wilcoxon test) for 10 min in response to 200 AP (40 Hz, 5 s) stimulation. Absolute line profiles of mDia1 overlapping with Bassoon (presynapse) distribution were integrated. Data shown are normalized to DMSO (set to 100) and expressed as mean ± SEM. N=3 independent experiments from n_DMSO = 92 synapses, n_Dynasore = 135 synapses. (I) Presynaptic mDia1 levels in synapses from hippocampal neurons transduced with wild-type Dynamin1 (WT; 100±6.2) or GTPase-deficient Dynamin1 (K44A; 142.9±8.3, p<0.0001, one sample Wilcoxon test) in response to 200 AP (40 Hz, 5 s) stimulation. Line profiles of mDia1 overlapping with Bassoon distribution were integrated. Data shown are normalized to Dynamin1-WT (set to 100) and expressed as mean ± SEM. N=2 independent experiments from n_WT = 43 synapses, n_K44A = 51 synapses.

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Figure 3—source data 2 Original scan for the anti-mDia1 and anti-Dynamin1 immunoblots from Figure 3F.: https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-data2-v2.zip
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Figure 3—source data 3 Original scan for the anti-Actin immunoblot from Figure 3F.: https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-data3-v2.zip
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Figure 3—source data 4 Original scans for immunoblots from Figure 3F with highlighted bands and sample labels.: https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-data4-v2.zip
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Based on these results we next investigated the relationship between mDia1/3 and presynaptic actin. Multicolor gSTED imaging confirmed that F-actin labeled by phalloidin was highly abundant at the postsynaptic compartment. In addition, F-actin was also visible, albeit at reduced levels, in presynaptic boutons marked by Bassoon (Figure 4A and B). Inhibition of Dynamin in the presence of Dynasore (Figure 4C and Figure 4—figure supplement 1A) or expression of Dynamin1 K44A (Figure 4—figure supplement 1B and C) significantly elevated presynaptic F-actin levels within the Bassoon area. These data are consistent with a role of presynaptic actin in SV endocytosis. We directly tested the role of mDia in presynaptic actin nucleation and actin-dependent SV endocytosis in several ways. First, we generated a mutant version of mDia1 that lacks the ability to nucleate F-actin due to site-specific inactivation of its FH2 domain (Daou et al., 2014; Higashi et al., 2008; Xu et al., 2004). We found that actin polymerization-defective mutant mDia1 (K994A) failed to restore delayed vGLUT1-pHluorin endocytosis in mDia1-depleted hippocampal neurons (Figure 4D and Figure 4—figure supplement 1D). Second, we analyzed the effects of depleting hippocampal neurons of mDia1/3 on presynaptic F-actin. We found that loss of mDia1/3 significantly reduced the amount of detectable F-actin within the Bassoon area (Figure 4A and E). To unequivocally distinguish between presynaptic actin at exo-/ endocytic sites and the abundant pool of postsynaptic actin associated with the postsynaptic density, we used the ORANGE strategy for CRISPR-based genome-engineering (Willems et al., 2020) to generate eGFP-Actb−knock-in (eGFP-β-actin KI) hippocampal neurons. Due to the low knock-in efficiency, this strategy allowed us to visualize endogenously tagged eGFP-β-actin by 2-color gSTED imaging exclusively in presynaptic boutons marked by vGLUT1. This assay confirmed the significant reduction in endogenous β-actin at presynapses from mDia1/3-depleted neurons (Figure 4F and G). Third, we reasoned that if loss of presynaptic actin in mDia-depleted neurons was causal for the observed defect in SV endocytosis, pharmacological stabilization of F-actin might rescue the endocytic phenotype. Treatment of mDia1/3-depleted neurons with jasplakinolide indeed fully restored the kinetics of SV endocytosis monitored by vGLUT1-pHluorin (Figure 4H and Figure 4—figure supplement 1H) and rescued presynaptic F-actin levels to that of controls (actin levels normalized to shCTR + DMSO, set to 100: shCTR + DMSO = 100 ± 6.3; shmDia1+3 + DMSO=47.7 ± 4.3; shCTR + Jasp = 150.6 ± 11.9; shmDia1+3 + Jasp=94.3 ± 11.5) (Figure 4—figure supplement 1G).

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mDia facilitates synaptic vesicle (SV) endocytosis by regulating presynaptic F-actin.

(A) Representative three-channel time-gated stimulated emission depletion (STED) images of synapses from hippocampal cultures transduced with *shCTR* or *shmDia1 +3*, targeting *Diaph1/2* genes, fixed and immunostained for Bassoon (presynaptic marker, magenta), F-Actin (cyan) and Homer1 (postsynaptic marker, green). Scale bar, 250 nm. (B) Averaged normalized line profiles for synaptic distribution of F-Actin and Homer1 relative to Bassoon (Maximum set to 0 nm). Data are expressed as mean ± SEM. N=4 independent experiments from n=154 synapses. (C) Presynaptic F-Actin levels in synapses treated with 0.1% dimethyl sulfoxide (DMSO) (100±4.8) or 80 µM Dynasore (134.7±6.8; p=0.001, one sample Wilcoxon test) for 10 min before fixation (Representative images in Figure 4—figure supplement 1A). Cells were immunostained for Bassoon (magenta), F-Actin (cyan), and Homer1 (green). Absolute line profiles of F-Actin overlapping with Bassoon (presynapse) distribution were integrated. Data shown are normalized to DMSO (set to 100) and expressed as mean ± SEM. N=3 independent experiments from n_DMSO = 207 synapses, n_Dynasore = 211 synapses. (D) Endocytic decay constants of vesicular glutamate transporter 1 (vGLUT1)-pHluorin traces from hippocampal neurons transduced with lentiviral particles encoding *shCTR* (τ_shCTR = 20.7 ± 0.9 s) or *shmDia1* (τ_shmDia1 = 26.4±2.0 s) in response to 200 action potential (AP) (40 Hz, 5 s) stimulation. For rescue experiments, neurons were co-transduced with lentiviruses encoding mDia1-WT-SNAP (τ_{shmDia1 + mDia1-WT}=16.1±1.9 s) or mDia1-K994A-SNAP (τ_{shmDia1 + mDia1-K994A}=29.0±1.9 s) (p_{shmDia1 vs shmDia1 + mDia1-WT}<0.01; p_{shmDia1 + mDia1-WT vs shmDia1 + mDia1-K994A}<0.001, one-way ANOVA with Tukey’s post-test). Data are expressed as mean ± SEM. N=6 independent experiments from n_shCTR = 21 videos; n_shmDia1=21 videos, n_{shmDia1 + mDia1-WT}=16 videos, n_{shmDia1 + mDia1-K994A}=19 videos. (E) Presynaptic F-Actin levels in synapses from hippocampal cultures transduced with *shCTR* (100±6.4) or *shmDia1 +3* (58.1±2.9; p<0.001, one sample Wilcoxon test). Line profiles of F-Actin overlapping with Bassoon (presynapse) distribution were integrated. Data shown are normalized to shCTR (set to 100) and expressed as mean ± SEM. N=4 independent experiments from n_shCTR = 155 synapses, n_shmDia1+3 = 158 synapses. (F) Representative confocal and two-channel time-gated STED images of endogenous β-Actin (cyan) in vGLUT1 (magenta) positive boutons from hippocampal neurons transduced with lentiviruses encoding *shCTR* or *shmDia1 +3*. Scale bar, 250 nm. (G) Analysis of presynaptic endogenous β-Actin levels in vGLUT1 positive boutons from *shCTR* (100±6.3) and *shmDia1 +3* (47.7±4.3; p<0.0001 one-sample Wilcoxon test) transduced neurons. β-Actin STED mean intensity was measured using a confocal vGLUT1 signal as a mask. Data shown are normalized to shCTR (set to 100) and expressed as mean ± SEM from two independent experiments and n_shCTR = 67 synapses, n_shmDia1+3 = 53 synapses. (H) Endocytic decay constants of vGLUT1-pHluorin traces (Figure 4—figure supplement 1H) for neurons transduced with *shCTR* or *shmDia1 +3* in response to 40 AP (20 Hz, 2 s) stimulation. Neurons were pre-incubated with 0.1% DMSO or 1 µM Jasplakinolide (Jasp) for 30 min in the media before imaging (τ_{shCTR + DMSO} = 13.4±1.0 s, τ_{shCTR + Jasp} = 15.0±2.2 s, τ_{shmDia1+3 + DMSO}=25.0±2.7 s, τ_{shmDia1+3 + Jasp}=15.6±2.4 s; p_{shCTR vs shmDia1+3} < 0.01; p_{shmDia1+3 + DMSO vs shmDia1+3 + Jasp}<0.05, one-way ANOVA with Tukey’s post-test). Data are expressed as mean ± SEM. N=6 independent experiments from n_{shCTR + DMSO} = 32 videos, n_{shmDia1+3 + DMSO}=35 videos, n_{shCTR + Jasp} = 33 videos; n_{shmDia1+3 + Jasp}=34 videos.

Figure 4—source data 1 Numerical source data for Figure 4B, C, D, E, G and H.: https://cdn.elifesciences.org/articles/92755/elife-92755-fig4-data1-v2.zip
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We conclude that mDia1/3 facilitates SV endocytosis by regulating presynaptic F-actin.

mDia1/3-Rho and Rac1 signaling pathways cooperatively act to facilitate presynaptic endocytosis

In non-neuronal cells, mDia proteins have been shown to be disinhibited by active Rho family GTPases, in particular RhoA (Otomo et al., 2005; Figure 5A and Figure 1—figure supplement 1N). We reasoned that Rho proteins might serve a similar function at presynaptic nerve terminals in hippocampal neurons. Multicolor gSTED imaging revealed that endogenous RhoA was mostly located within the presynaptic compartment (Figure 5B and C). Interference with Rho function by co-expression of dominant-negative (DN) variants of RhoA and RhoB (Figure 5D and Figure 5—figure supplement 1A) or co-depletion of these Rho isoforms, by shRNA-mediated targeting of Rhoa and Rhob genes (shRhoA +B; Figure 5—figure supplement 1C), delayed SV endocytosis in response to stimulation with 200 APs without impacting the apparent levels of exocytosis (Figure 5—figure supplement 1B and D). These results are consistent with a model in which active Rho promotes mDia function and, thereby F-actin nucleation.

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RhoA/B facilitates presynaptic endocytosis and are regulated by mDia1/3.

(A) Schematic representation of activation of mDia1 by RhoA-GTP and positive feedback loop of mDia1 on RhoA-GTP levels through GEF stimulation. (B) Representative three-channel time-gated STED image of synapses from hippocampal cultures, fixed and immunostained for Bassoon (magenta), RhoA (cyan), and Homer1 (green). Scale bar, 250 nm. (C) Averaged normalized line profiles for synaptic distribution of RhoA and Homer1 relative to Bassoon (Maximum set to 0 nm). Data are expressed as mean ± SEM. N=5 independent experiments from n=230 synapses. (D) Endocytic decay constants of averaged normalized Synaptophysin-pHluorin fluorescence traces (Figure 5—figure supplement 1A) in response to 200 action potential (AP) (40 Hz, 5 s) stimulation. Neurons were transfected with the annotated combinations of plasmids encoding wild-type (WT) or dominant-negative (DN, T19N mutation) RhoA and RhoB (τ_{RhoA-WT + RhoB-WT}=18.4±0.7 s, τ_{RhoA-WT + RhoB-DN} = 16.0±1.0 s, τ_{RhoA-DN + RhoB-WT}=19.8±2.4 s, τ_{RhoA-DN + RhoB-DN}=30.1±1.0 s; p_{RhoA-WT + RhoB-WT vs RhoA-DN + RhoB-DN}<0.01, one-way ANOVA with Tukey’s post-test). Data shown represent mean ± SEM. N=3 independent experiments from n_{RhoA-WT + RhoB-WT}=21 videos, n_{RhoA-DN + RhoB-WT}=31 videos, n_{RhoA-WT + RhoB-DN}=23 videos, n_{RhoA-DN + RhoB-DN}=22 videos. (E) Analysis of RhoA activity by RhoA-GTP pulldown (PD) from whole-cell lysates (input) of mouse hippocampal neurons expressing shCTR or shmDia1 +3 using immobilized Rhotekin as a bait. Samples were analyzed by immunoblotting for mDia1, mDia3, RhoA, and Tubulin using specific antibodies. Input, 10% of material used for the pulldown. The contrast of pulldown and input blots was seperately adjusted for visualization purposes. (F) Densitometric quantification of RhoA-GTP normalized to total RhoA levels (input) in lysates from neurons transduced with shCTR or shmDia1 +3 (0.7±0.0, p<0.001, one sample t-test) from immunoblots exemplified in E. Values for shCTR were set to 1. Data are expressed as mean ± SEM from N=3 independent experiments.

Figure 5—source data 1 Numerical source data for Figure 5C, D and F.: https://cdn.elifesciences.org/articles/92755/elife-92755-fig5-data1-v2.zip
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Figure 5—source data 2 Original scans for the anti-mDia1, anti-Tubulin, anti-RhoA, and anti-mDia3 immunoblots from Figure 5E.: https://cdn.elifesciences.org/articles/92755/elife-92755-fig5-data2-v2.zip
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Figure 5—source data 3 Original scans for immunoblots from Figure 5E with highlighted bands and sample labels.: https://cdn.elifesciences.org/articles/92755/elife-92755-fig5-data3-v2.zip
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Figure 5—source data 4 Original scans for the anti-RhoA immunoblots used for analysis are shown in Figure 5F.: https://cdn.elifesciences.org/articles/92755/elife-92755-fig5-data4-v2.zip
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Figure 5—source data 5 Original scans for immunoblots used for analysis are shown in Figure 5F with highlighted bands and sample labels.: https://cdn.elifesciences.org/articles/92755/elife-92755-fig5-data5-v2.zip
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F-actin dynamics are known to be controlled by interdependent signaling networks characterized by feedback regulation between components within the same pathway and between key regulatory switches (e.g. individual Rho/Rac family members) that drive distinct forms of F-actin (see below) (Lawson and Ridley, 2018). For example, active mDia is known to stimulate RhoA activation (Kitzing et al., 2007; Figure 5A). Consistently, we found that RhoA activity was reduced in hippocampal neurons depleted of mDia1/3 (Figure 5E and F). Work in non-neuronal cells has further revealed that RhoA activity antagonizes activation of Rac1 (Chauhan et al., 2011), a key factor for promoting the formation of branched F-actin networks at the cell cortex (Hodge and Ridley, 2016; Lawson and Ridley, 2018; Figure 6A). We hypothesized that reciprocally interdependent Rho/ Rac1 signaling might control presynaptic F-actin assembly and, thereby, SV endocytosis. In agreement with the alleged antagonistic regulation of RhoA and Rac1 function (Lawson and Ridley, 2018), we found Rac1 activity to be significantly elevated in hippocampal neurons depleted of mDia1/3 (Figure 6B and C), e.g., under conditions of reduced Rho-GTP levels. A similar increase in active Rac1 levels was observed upon pharmacological inhibition of RhoA/B in the presence of Rhosin (Figure 6—figure supplement 1A). Multicolor gSTED imaging showed that Rac1 was equally distributed between pre- and postsynaptic compartments in unperturbed hippocampal neurons (Figure 6D and E). Elevated active Rac1 levels might conceivably ameliorate presynaptic endocytic phenotypes elicted by mDia1/3 loss. In support of this hypothesis, we found that selective pharmacological inhibition of Rac1 reduced presynaptic actin levels (Figure 6—figure supplement 1B and C) and caused a delay in the endocytic retrieval of endogenous vGAT (Figure 6F and G). Moreover, Rac1 inhibition appeared to further aggravate impaired vGAT endocytosis in mDia1/3-depleted neurons, although the effect remained below statistical significance. Expression of constitutively active GTP-locked Rac1 (Rac1-CA) restored normal Syph-pHluorin endocytosis kinetics in mDia1/3-depleted neurons, whereas Syph-pHluorin endocytosis was delayed upon expression of dominant-negative Rac1 (Rac1-DN) and exacerbated the endocytic phenotype of mDia1/3 loss (Figure 6H and Figure 6—figure supplement 1E). No overt effect of Rac1 inhibition or overexpression of either Rac1 form on SV exocytosis was observed (Figure 6—figure supplement 1D and F). Perturbation of the related Cdc42 protein, another actin regulatory GTPase Lawson and Ridley, 2018 found at synapses (Figure 6—figure supplement 1G–I), did not significantly affect the kinetics of SV endocytosis in control or mDia1/3-depleted neurons (Figure 6—figure supplement 1J and K). These data suggest that interdependent mDia1/3 and Rac1-based pathways control SV endocytosis via presynaptic actin.

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mDia1/3-Rho and Rac1 signaling facilitates presynaptic endocytosis.

(A) Schematic of the interplay between RhoA and Rac1 signaling via GTPase regulatory proteins (e.g. GTPase activating proteins (GAPs) among others) common for RhoA and Rac1. (B) Analysis of Rac1 activity by Rac1-GTP pulldown (PD) from whole-cell lysates (input) of mouse hippocampal neurons expressing shCTR or shmDia1 +3 utilizing immobilized PAK as a bait. Samples were analyzed by immunoblotting for mDia1, mDia3, Rac1, and Tubulin using specific antibodies. Input, 10% of material used for the pulldown. The contrast of pulldown and input blots was seperately adjusted for visualization purposes. (C) Densitometric quantification of Rac1-GTP normalized to total Rac1 levels (input) in lysates from neurons transduced with shCTR or shmDia1 +3 (2.2±0.2; p<0.05, one sample t-test) from immunoblots exemplified in (B). Values for shCTR were set to 1. Data are expressed as mean ± SEM from N=3 independent experiments. (D) Representative three-channel time-gated stimulated emission depletion (STED) image of synapses from hippocampal cultures, fixed and immunostained for Bassoon (magenta), Rac1 (cyan), and Homer1 (green). Scale bar, 250 nm. (E) Averaged normalized line profiles for synaptic distribution of Rac1 and Homer1 relative to Bassoon (Maximum set to 0 nm). Data represent mean ± SEM. N=3 independent experiments from n=79 synapses. (F) Averaged normalized vGAT-CypHer fluorescence traces for neurons transduced with *shCTR* or *shmDia1 +3* in response to 200 AP (40 Hz, 5 s) stimulation. Cells were acutely treated with 0.1% DMSO or 10 µM Rac1 Inhibitor (EHT 1864) in the imaging buffer. Data shown represent the mean ± SEM. N=8 independent experiments from n_{shCTR + DMSO} = 46 videos, n_{shmDia1+3 + DMSO} = 45 videos, n_{shCTR + EHT 1864} = 42 videos, n_{shmDia1+3 + EHT 1864} = 43 videos. (G) Endocytic decay constants of vGAT-CypHer traces in F: τ_{shCTR + DMSO} = 14.7±0.9 s, τ_{shmDia1+3 + DMSO}=27.5±2.3 s, τ_{shCTR + EHT 1864} = 30.3±6.7 s, τ_{shmDia1+3 + EHT 1864} = 41.0±4.3 s; p_{shCTR + DMSO vs shmDia1+3 + DMSO}<0.05, p_{shCTR + DMSO vs shmDia1+3 + EHT 1864} < 0.0001, Kruskal-Wallis test with Dunn’s post-test. Data represent mean ± SEM. (H) Endocytic decay constants of Synaptophysin-pHluorin traces (Figure 6—figure supplement 1E) of neurons transduced with *shCTR* (τ_shCTR = 12.0±0.7 s) or *shmDia1 +3* (τ_shmDia1+3 = 22.7±2.0 s) and transfected with constitutively active Rac1 (Rac1-CA; Q61L variant; τ_{shCTR + Rac1-CA}=13.6±1.2 s, τ_{shmDia1+3 + Rac1-CA}=13.3±1.4 s) or dominant negative Rac1 (Rac1-DN; T17N variant; τ_{shCTR + Rac1-DN} = 27.8±1.3 s, τ_{shmDia1+3 + Rac1-DN} = 33.4±1.6 s) in response to 200 AP (40 Hz, 5 s) stimulation (p_{shCTR vs shmDia1+3} < 0.01; p_{shCTR vs shCTR + Rac1-DN}<0.0001, p_{shCTR vs shmDia1+3 + Rac1-DN}<0.01, p_{shmDia1+3 vs shmDia1+3 + Rac1-DN}<0.01, one-way ANOVA with Tukey’s post-test). Data are expressed as mean ± SEM. N=3 independent experiments from n_shCTR = 12 videos, n_shmDia1+3 = 23 videos; n_{shCTR + Rac1-CA}=10 videos, n_{shmDia1+3 + Rac1-CA}=14 videos, n_{shCTR + Rac1-DN} = 9 videos; n_{shmDia1+3 + Rac1-DN} = 13 videos.

Figure 6—source data 1 Original scans for the anti-mDia3, anti-Tubulin, and anti-Rac1 immunoblots from Figure 6B.: https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-data1-v2.zip
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Figure 6—source data 2 Original scan for the anti-mDia1 immunoblot from Figure 6B.: https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-data2-v2.zip
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Figure 6—source data 3 Original scans for immunoblots from Figure 6B with highlighted bands and sample labels.: https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-data3-v2.zip
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Figure 6—source data 4 Numerical source data for Figure 6C, E, F, G and H.: https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-data4-v2.zip
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Figure 6—source data 5 Original scans for the anti-Rac1 immunoblots used for analysis are shown in Figure 6C.: https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-data5-v2.zip
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Figure 6—source data 6 Original scans for immunoblots used for analysis are shown in Figure 6C with highlighted bands and sample labels.: https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-data6-v2.zip
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We probed this model by analyzing the ultrastructure of synapses from hippocampal neurons depleted of mDia1/3 or following inhibition of Rac1 activity. Synapses from mDia1/3-depleted neurons displayed an accumulation of non-coated plasma membrane invaginations that were found both in the vicinity of the active zone as well as distal from the synaptic contact area (Figure 7A and B). Moreover, we observed elevated numbers of endosome-like vacuoles (ELVs) (Figure 7A and C) that might serve as a donor membrane for SV reformation (Kononenko and Haucke, 2015; Kononenko et al., 2014; Watanabe et al., 2014) as suggested by the observed reduction in SV numbers in mDia-depleted neurons (compare Figure 2). A prominent accumulation of plasma membrane invaginations (Figure 7—figure supplement 1A) and endosome-like vacuoles (Figure 7—figure supplement 1B) was similarly found in hippocampal neurons from mDia1 KO mice. Inhibition of Rac1 function by EHT 1864 also led to the accumulation of non-coated plasma membrane invaginations (Figure 7A and D), whereas the number of endosome-like vacuoles was not significantly altered (Figure 7A and E). Pharmacological blockade of Rac1 activity in neurons depleted of mDia1/3 further increased the number of plasma membrane invaginations significantly, while the additional effect on the number of endosome-like vacuoles remained insignificant (Figure 7F, G and H). mDia1/3 loss and Rac1 perturbation exhibited similar phenotypes with respect to the length and width of tubular membrane invaginations (Figure 7—figure supplement 1C and D).

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Defects in presynaptic ultrastructure induced by interference with mDia1/3-Rho and Rac1 signaling.

(A) Representative synaptic electron micrographs from hippocampal neurons transduced with lentiviruses encoding *shCTR* or *shmDia1 +3*, targeting *Diaph1/2* genes, and treated with 0.1% dimethyl sulfoxide (DMSO) or 10 µM Rac1 Inhibitor (EHT 1864) for 2 hr before fixation. Invaginations and endosome-like vacuoles (ELVs) are colored in blue and yellow, while postsynapse and synaptic cleft are colored in green and maroon, respectively. Scale bar, 250 nm. (B) Average number of invaginations per μm² in *shCTR* (0.5±0.1) and *shmDia1 +3* (0.9±0.1; p<0.0001, Mann-Whitney test) boutons. Data represent mean ± SEM. N=3 independent experiments from n_shCTR = 326 synapses, n_shmDia1+3 = 323 synapses. (C) Average number of ELVs per μm² in shCTR (1.4±0.1) and *shmDia1 +*3 (2.7±0.2; p<0.0001, Mann-Whitney test) boutons. Data represent mean ± SEM. N=3 independent experiments from n_shCTR = 326 synapses, n_shmDia1+3 = 323 synapses. (D) Average number of invaginations per μm² in *shCTR* and *shmDia1 +3* boutons treated with 0.1% DMSO (0.8±0.1 for *shCTR*; 1.2±0.1 for *shmDia1 +3*, p_{shCTR + DMSO vs shmDia1+3 + DMSO}<0.01) or 10 µM EHT 1864 (1.8±0.1 for *shCTR*, p_{shCTR + DMSO vs shCTR + EHT 1864} < 0.0001; 1.9±0.2 for *shmDia1 +3*, p_{shCTR + DMSO vs shmDia1+3 + EHT 1864} < 0.0001, p_{shmDia1+3 + DMSO vs shmDia1+3 + EHT 1864} < 0.05, Kruskal-Wallis test with Dunn’s post-test) for 2 hr before fixation. Data represent mean ± SEM from n_{shCTR + DMSO} = 144 synapses, n_{shmDia1+3 + DMSO}=143 synapses, n_{shCTR + EHT 1864} = 136 synapses, n_{shmDia1+3 + EHT 1864} = 153 synapses. (E) Average number of ELVs per μm² in *shCTR* and *shmDia1 +3* boutons treated with 0.1% DMSO (2.2±0.2 for *shCTR*; 3.3±0.3 for *shmDia1 +3*, p_{shCTR + DMSO vs shmDia1+3 + DMSO}<0.05) or 10 µM EHT 1864 (2.6±0.3 for *shCTR*; 4.3±0.4 for *shmDia1 +3*, p_{shCTR + DMSO vs shmDia1+3 + EHT 1864} < 0.001, p_{shCTR + EHT 1864 vs shmDia1+3 + EHT 1864} < 0.01, Kruskal-Wallis test with Dunn’s post-test) for 2 hr before fixation. Data represent mean ± SEM from n_{shCTR + DMSO} = 144 synapses, n_{shmDia1+3 + DMSO}=143 synapses, n_{shCTR + EHT 1864} = 136 synapses, n_{shmDia1+3 + EHT 1864} = 153 synapses.

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Collectively, these findings demonstrate that mDia1/3-Rho and Rac1 signaling pathways cooperatively act to facilitate presynaptic endocytosis and SV recycling.

Discussion

We found that loss of mDia1 either alone or in conjunction with the closely related mDia3 isoform slows the kinetics of SV endocytosis of exogenous pHluorin-tagged SV proteins and of endogenous vGAT without perturbing exocytic SV fusion. These phenotypes are accompanied by the accumulation of plasma membrane invaginations and vacuolar structures as well as an activity-dependent reduction of the total SV pool (Figures 1, 2 and 7). Furthermore, we observe that interdependent mDia1/3-Rho and Rac1 signaling pathways cooperatively act to facilitate presynaptic endocytosis (Figures 5—7). Several lines of evidence indicate that these endocytic phenotypes are a consequence of perturbed presynaptic actin levels or dynamics. (i) Endogenous presynaptic F-actin is reduced in mDia1/3-depleted hippocampal synapses (Figure 4) and (ii) actin polymerization-defective mDia1 fails to restore normal endocytosis kinetics in hippocampal neurons depleted of mDia1 (Figure 4). Conversely, (iii) pharmacological stabilization of F-actin by jasplakinolide (Figure 4) or (iv) expression of constitutively-active Rac1 to drive compensatory actin polymerization (for example via other formins and/ or ARP2/3) (Figure 6) rescues endocytosis in mDia1/3-KD hippocampal neurons. Moreover, (v) we find mDia1 (Figure 3) and F-actin (Figure 4) to accumulate at presynapses under conditions of impaired dynamin-dependent endocytosis. Together with the observation that inhibition of F-actin dynamics (Peng et al., 2011) in the combined presence of latrunculin A, jasplakinolide, and Y-27632 (Figure 1) slows the kinetics of SV endocytosis, our data support the hypothesis that presynaptic actin facilitates SV endocytosis downstream of mDia/ RhoA and Rac1-based signaling pathways.

Our data extend previous studies using conditional genetics that have identified an actin requirement for SV endocytosis (Wu et al., 2016). The endocytic phenotype described here for loss of mDia1/3, however, is substantially milder than that elicited by complete loss of β− or γ-actin, likely reflecting the fact that several partially redundant pathways cooperate to promote F-actin assembly at synapses (see below). In contrast to Actb or Actg1 KO hippocampal neurons (Wu et al., 2016) we did not observe strong exocytic depression in response to train stimulation in mDia-depleted neurons (e.g. Figure 1—figure supplement 1A–M) although we cannot rule out mild differences in synaptic strength and/ or release probability as observed in Rac1 KO hippocampal neurons and at the calyx of Held (Keine et al., 2022; O’Neil et al., 2021).

At the ultrastructural level, we find that genetic (KO) or lentiviral induced (KD) loss of the genes Diaph1 or Diaph1/2, encoding mDia1 or mDia1/3, respectively, partially phenocopies acute perturbation of formin-mediated actin assembly (Soykan et al., 2017) with respect to the accumulation of plasma membrane invaginations. Unlike acute formin inhibition (Soykan et al., 2017), we find elevated numbers of post-fission endosome-like vacuoles in mDia-depleted hippocampal synapses. The latter correlates with a mild reduction of SV numbers, suggesting that mDia may have a further role in SV reformation downstream of the actual endocytosis reaction (Chanaday et al., 2019; Gan and Watanabe, 2018; Kononenko et al., 2014). The identified function of mDia1/3-mediated presynaptic actin assembly in presynaptic endocytosis and SV recycling is consistent with the actin-binding properties of many endocytic proteins including Dynamin (Ferguson et al., 2009; Gu et al., 2014), PACSINs (Kessels and Qualmann, 2006), or Epsins, among others (McMahon and Boucrot, 2011; Merrifield et al., 2005; Saheki and De Camilli, 2012). How exactly actin functions to facilitate endocytosis of SV membranes remains to be fully understood. The comparably small size of the membrane invaginations that accumulate following pharmacological inhibition of formins (<100 nm) or Rac1 or upon loss of mDia1 (typically <150 nm, although larger ones are observed) render an actomyosin-based constriction mechanism for SV endocytosis unlikely (consistent with Sankaranarayanan et al., 2003). We rather favor a - possibly indirect - function of F-actin in presynaptic endocytosis via the formation of an F-actin ring that couples exocytic membrane compression to endocytic pit formation (Ogunmowo et al., 2023). Such a model is supported by the abundance of large plasma membrane invaginations distal from the active zone where SV fusion occurs at synapses following mDia1/3 and Rac1 loss of function (Figure 7). Consistent with this, it has been shown that mDia1 controls presynaptic membrane contractility (Deguchi et al., 2016), e.g., via the recently identified presynaptic actin corrals visualized in genome-engineered rat hippocampal neurons (Bingham et al., 2023).

A key result from our study is the finding that presynaptic endocytosis is regulated by interdependent, yet partially redundant signaling for F-actin assembly downstream of Rho and Rac1, e.g., pathways known to control linear and branched F-actin networks (Lawson and Ridley, 2018; Müller et al., 2020) that were shown to co-exist within hippocampal presynaptic boutons (Bingham et al., 2023). We demonstrate that loss of mDia1/3 or inhibition of Rho causes the hyperactivation of the actin-promoting GTPase Rac1 and that dual loss of mDia1/3 loss and Rac1 function results in synergistic inhibitory effects with respect to presynaptic endocytosis and ultrastructural defects (Figures 5—7). These findings suggest that the signaling network that regulates presynaptic actin assembly may have been evolutionarily selected for plasticity and resilience. A resilient network for the control of actin dynamics at the presynapse may also help to explain the partially contradicting results from drug-based manipulations of actin and actin regulatory factors in different models of presynaptic neurotransmission reported in the past (Bleckert et al., 2012; Del Signore et al., 2021; Hori et al., 2022; Piriya Ananda Babu et al., 2020; Richards et al., 2004; Sankaranarayanan et al., 2003; Shupliakov et al., 2002; Wu and Chan, 2022; Wu et al., 2016). Moreover, accumulating evidence indicates that different pools of actin might be differentially amenable to drug treatments (Bleckert et al., 2012), e.g., as a consequence of the association of actin filaments with molecules such as tropomyosin (Gormal et al., 2017) that render them resistant to drugs, and/ or variations in the fraction of F-actin vs. monomeric actin pools (Higashida et al., 2008), providing a possible explanation for previously discrepant results.

A number of open questions remain. We predict that presynaptic actin assembly via mDia/Rho and Rac1 facilitates all forms of presynaptic endocytosis that operate on timescales of milliseconds to seconds (Chanaday and Kavalali, 2018; Clayton and Cousin, 2009; Watanabe and Boucrot, 2017) and at many types of synapses (e.g. small central synapses, ribbon synapses, the neuromuscular junction), but further studies will be needed to test this hypothesis. We note that fast endophilin-mediated endocytosis (FEME), a process with resemblance to endocytosis at synapses (Watanabe and Boucrot, 2017), has been shown to be differentially regulated by actin and Rho GTPase family members (Boucrot et al., 2015; Renard et al., 2015). At present we do not understand if and how RhoA/B and Rac1 GTPase activation is nested into the SV cycle and whether these GTPases operate at the same or different nanoscale sites and on the same or different membranes. Future studies will be required to tackle these questions.

Materials and methods

Materials

Animals

Primary neurons were obtained from either wild-type C57BL/6 J (Charles River, RRID: IMSR_JAX:000664) or Diaph1 KO mice (Peng et al., 2007; Peng et al., 2003). All animal experiments were reviewed and approved by the ethics committee of the Landesamt für Gesundheit und Soziales (LAGeSo) Berlin or the Committee on the Ethics of Animal Experiments of Columbia University and conducted according to the committees’ guidelines (LAGeSo) or the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (for Diaph1 KO mice). At the facilities, animal care officers monitored compliance with all regulations. Mice were group-housed under 12/12 hr light/dark cycle with access to food and water ad libitum. Mice from both genders were used and cultures were randomly allocated to experimental groups (e.g. different treatments). Multiple independent experiments using several biological replicates were carried out as indicated in the Figure legends.

Antibodies

Antibodies and their working dilutions used in this study are denoted in the Key Resource Table (IB: Immunoblot; IC: Immunocytochemistry; IP: Immunoprecipitation). Antibodies were stored according to the manufacturer’s recommendations. All secondary antibodies are species-specific (highly cross-adsorbed).

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (Mus musculus)	C57BL/6 N wild-type	Leibniz Research Institute for Molecular Pharmacology	RRID:IMSR_JAX:000664
Genetic reagent (Mus musculus)	mDia1 KO	Bartolini Lab, Columbia University	Peng et al., 2003; https://doi.org/10.1016/s0960-9822(03)00170–2	Genetic knockout of Diaph1
Cell line (Homo sapiens)	HEK293T	American Type Culture Collection	Cat# CRL-3216; RRID:CVCL_0063
Antibody	β-Actin (mouse monoclonal)	Sigma-Aldrich	Cat# A5441; RRID:AB_476744	IB (1:5000)
Antibody	Bassoon (guinea pig monoclonal)	Synaptic Systems	Cat# 141 318; RRID:AB_2927388	IC (1:100)
Antibody	CDC42 (rabbit polyclonal)	Abcam	Cat# ab64533; RRID:AB_1310067	IB (1:1000); IC (1:100)
Antibody	Dynamin1 (rabbit polyclonal)	Pietro D. Camilli	Shupliakov et al., 2002; DOI: 10.1126/science.276.5310.29	IB (1:2000)
Antibody	GFP (mouse monoclonal)	Thermo Fisher Scientific	Cat# A-11120; RRID:AB_221568	IC (1:2500)
Antibody	Homer1 (mouse monoclonal)	Synaptic Systems	Cat# 160 011; RRID:AB_2120992	IC (1:200)
Antibody	Homer1 (rabbit polyclonal)	Synaptic Systems	Cat# 160 003; RRID:AB_887730	IC (1:200)
Antibody	LAMP1 (rabbit monoclonal)	Cell Signaling Technology	Cat# 9091; RRID:AB_2687579	IB (1:1000)
Antibody	mDia1 (mouse monoclonal)	BD Biosciences	Cat# 610848; RRID:AB_398167	IB (1:500); IP (2 µg)
Antibody	mDia1 (rabbit monoclonal)	Abcam	Cat# ab129167; RRID:AB_11143749	IC (1:100)
Antibody	mDia3 (rabbit polyclonal)	Sigma-Aldrich	Cat# HPA005647; RRID:AB_1078657	IB (1:1000)
Antibody	Myosin IIb (mouse monoclonal)	Abcam	Cat# ab684; RRID:AB_305661	IC (1:100)
Antibody	Myosin IIb (rabbit polyclonal)	Cell Signaling Technology	Cat# 3404; RRID:AB_126421	IB (1:2000)
Antibody	Rac1 (mouse monoclonal)	BD Biosciences	Cat# 610650; RRID:AB_397977	IB (1:1000); IC (1:50)
Antibody	RFP (rabbit polyclonal)	Takara Bio	Cat# 632496; RRID:AB_10013483	IC (1:500)
Antibody	RhoA (rabbit monoclonal)	Cell Signaling Technology	Cat# 2117; RRID:AB_10693922	IB (1:1000); IC (1:100)
Antibody	α-Tubulin (mouse monoclonal)	Sigma-Aldrich	Cat# T9026; RRID:AB_47759	IB (1:5000)
Antibody	IgG control (mouse monoclonal)	Thermo Fisher Scientific	Cat# 31903; RRID:AB_10959891	IP (2 μg)
Antibody	vGAT-CypHer5E (rabbit polyclonal)	Synaptic Systems	Cat# 131 103CpH; RRID:AB_2189809	Live-imaging (1:500)
Antibody	Anti-mouse IgG Atto542 (donkey polyclonal)	Martin Lehmann	Gonschior et al., 2022; https://doi.org/10.1038/s41467-022-32533-4	IC (1:400)
Antibody	Anti-rabbit IgG Atto542 (donkey polyclonal)	Martin Lehmann	Gonschior et al., 2022; https://doi.org/10.1038/s41467-022-32533-4	IC (1:400)
Antibody	Anti-mouse Alexa Fluor 594 (goat polyclonal)	Thermo Fisher Scientific	Cat# A-11032; RRID:AB_2534091	IC (1:200)
Antibody	Anti-rabbit Alexa Fluor 594 (goat polyclonal)	Thermo Fisher Scientific	Cat# A-11037; RRID:AB_2534095	IC (1:200)
Antibody	Anti-guinea pig Atto647N (camelid monoclonal)	Synaptic Systems	Cat# N0602-At647N-S; RRID:AB_2744576	IC (1:200)
Antibody	Anti-mouse Alexa Fluor 647 (goat polyclonal)	Thermo Fisher Scientific	Cat# A-21235; RRID:AB_2535804	IC (1:200)
Antibody	Anti-mouse IRDye 680RD (goat polyclonal)	LI-COR Biosciences	Cat# 925–68070; RRID:AB_2651128	IB (1:10000)
Antibody	Anti-rabbit IRDye 680RD (goat polyclonal)	LI-COR Biosciences	Cat# 926–68071; RRID:AB_10956166	IB (1:10000)
Antibody	Anti-mouse IgG, IRDye 800CW (goat polyclonal)	LI-COR Biosciences	Cat# 926–32210; RRID:AB_621842	IB (1:10000)
Antibody	Anti-rabbit IgG, IRDye 800CW (goat polyclonal)	LI-COR Biosciences	Cat# 926–32211; RRID:AB_621843	IB (1:10000)
Chemical compound, drug	Phalloidin-Alexa Fluor 594	AAT Bioquest	Cat# ABD-23158	IC (1:1000)
Recombinant DNA reagent	Synaptophysin-pHluorin	Leon Lagnado	Gonschior et al., 2022; https://doi.org/10.1038/s41467-022-32533-4	Expresses rat Synaptophysin-2xpHluorin (inserted between Asn₁₈₃ – Thr₁₈₄) under a CMV promotor
Recombinant DNA reagent	vGlut1-pHluorin	Volker Haucke	Bolz et al., 2023; https://doi.org/10.1016/j.neuron.2023.08.016	Expresses rat vGlut1-pHluorin (inserted between Gly₉₉ – Gly₁₀₀) under a hSyn1 promotor; lentiviral plasmid
Recombinant DNA reagent	mCherry	Clontech	Cat# 632523	Expresses mCherry under a CMV promotor
Recombinant DNA reagent	mDia1-WT-mCherry	This study	-	Expresses mouse mDia1-WT-mCherry under a CMV promotor
Recombinant DNA reagent	mDia1-ΔN -mCherry	This study	-	Expresses truncation (first 60 AA) variant of mouse mDia1-mCherry under a CMV promotor
Recombinant DNA reagent	mDia1-WT-SNAP	This study	-	Expresses mouse mDia1-WT-SNAP under a hSyn1 promotor; lentiviral plasmid
Recombinant DNA reagent	mDia1-K994A-SNAP	This study	-	Expresses K994A variant of mDia1-SNAP under a hSyn1 promotor; lentiviral plasmid

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Actin dynamics and actin-nucleating mDia1/3 proteins facilitate synaptic vesicle (SV) endocytosis.

Figure 1—source data 1

Loss of mDia1/3 causes an activity-dependent reduction of the synaptic vesicle (SV) pool.

Figure 2—source data 1

mDia1 associates with endocytic proteins and localizes to presynaptic sites.

Figure 3—source data 1

Figure 3—source data 2

Figure 3—source data 3

Figure 3—source data 4

mDia facilitates synaptic vesicle (SV) endocytosis by regulating presynaptic F-actin.

Figure 4—source data 1

RhoA/B facilitates presynaptic endocytosis and are regulated by mDia1/3.

Figure 5—source data 1

Figure 5—source data 2

Figure 5—source data 3

Figure 5—source data 4

Figure 5—source data 5

mDia1/3-Rho and Rac1 signaling facilitates presynaptic endocytosis.

Figure 6—source data 1

Figure 6—source data 2

Figure 6—source data 3

Figure 6—source data 4

Figure 6—source data 5

Figure 6—source data 6

Defects in presynaptic ultrastructure induced by interference with mDia1/3-Rho and Rac1 signaling.

Figure 7—source data 1

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Kristine Oevel

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Svea Hohensee

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Atul Kumar

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Irving Rosas-Brugada (deceased)

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Francesca Bartolini

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Tolga Soykan

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Volker Haucke

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