CHAPTER 4

Munc18-1 regulates the cortical F-actin network inchromaffin cells

Julia Kurps1, Jan R.T. van Weering2, Matthijs Verhage1,2

1Department of Functional Genomics (VU)2Department of Clinical Genetics (VUmc)

Center for Neurogenomics and Cognitive Research

VU University Amsterdam and VU Medical Center

1081 HV Amsterdam, The Netherlands

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Abstract

The cortical F-actin network in adrenal chromaffin cells was proposed to be both abarrier and a facilitator for secretory vesicle trafficking and fusion and is reported tobe increased in secretion-deficient cells lacking Munc18-1. To investigate this effectof Munc18-1 deficiency on the actin network, we expressed Munc18-1, Munc18-1 or-thologues and Munc18-1 mutants in munc18-1 null CCs and analyzed effects on thecortical F-actin and Syntaxin1a distribution. Novel, automated analysis showed thatboth the total F-actin immunoreactivity (staining intensity) and the thickness of thenetwork were increased in munc18-1 null cells, but not in other secretion deficient mu-tants, such as syt-1 null and snap-25 null. All Munc18 orthologues, UNC18, mVps33aand mVps45, and most Munc18-1 mutants rescued cortical F-actin similar to wild typeMunc18-1. All Munc18 orthologues and mutants rescued Syntaxin1a targeting to thePM at least as well as wild type Munc18-1. We identified one mutant (Munc18V263T)that rescued the Syntaxin1a phenotype, but not the cortical F-actin phenotype. Thisfinding, together with homology and alignment studies suggests that hydrophobicity atthis position is crucial for the Munc18-1 dependent cortical F-actin regulation.

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Introduction

Actin is abundant in all eukaryotic cells and involved in several essential cellular pro-cesses. In neurons, actin plays a crucial role in developmental processes, morphologicalstability and motility. Actin filaments are also involved in slow axonal transport pro-cesses in mature neurons (Morris and Hollenbeck, 1995). The presence of actin inpre-synapses was suggested to regulate neurotransmitter release (Morales et al., 2000;Colicos et al., 2001). However, the role of synaptic actin in regulated exocytosis is stillstrongly debated.

Adrenal CCs have a dense cortical network of F-actin that supports the PM. Actinfilaments form tracks on which secretory vesicles are transported to the PM (Lang et al.,2000). Stimulation-dependent de-polymerization of actin filaments was suggested tofacilitate regulated exocytosis in CCs (Vitale et al., 1991; Trifaro et al., 1992). TheF-actin network is dynamic and it requires stimulus-dependent spatial and temporalregulation. The main mechanism of actin dynamics is actin-tread-milling (Carlier andPantaloni, 1997). During this process actin-monomers are added to the barbed (plus)end of existing actin fibers and actin-bound ATP is converted to ADP. The removalof ADP-bound actin monomers only occurs at the pointed (minus) end of actin fibers.The functional interplay of those two processes guarantees the directionality of actinfibers, which is essential for trafficking of secretory vesicles to the PM or cell motility ingeneral. Besides tread-milling, a multitude of other actin-mediating processes is known,e.g., branching, cross-linking, capping and severing (for review see (Lee and Dominguez,2010; Villanueva et al., 2012). A variety of actin-regulating proteins and their functionsin specific processes has been described extensively (see general introduction of thisthesis).

It was shown that the Sec1/Munc18 (SM) protein Munc18-1, which has an essen-tial function in docking and fusion of secretory vesicles in adrenal CCs (Voets et al.,2001), has an additional role in the regulation of the cortical F-actin network (Toonenet al., 2006). The absence of Munc18-1 in CCs resulted in an expansion of the subplas-malemmal F-actin network (Toonen et al., 2006). The underlying molecular mechanismremained unclear, at least partly due to the difficulty in quantifying the observed alter-ations. We applied an automated image analysis tool (PlasMACC (Kurps et al., 2014),Chapter 3 of this thesis) to further investigate the relationship between Munc18-1 andthe cortical F-actin network. We expressed members of the SM protein family andseveral Munc18-1 mutants (e.g., phospho-mutants and point mutations of highly con-served residues) in munc18-1 null MCCs and analyzed whether their effect on F-actinwas conserved. We found that all members of the SM protein family and most Munc18-1 mutants have the ability to regulate F-actin. We found one Munc18-1 mutant thatis unable to rescue the F-actin phenotype in munc18-1 null MCCs. This mutant willbe of importance to study the interaction between Munc18-1 and the cortical F-actinnetwork in adrenal CCs.

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Results

The cortical F-actin network is altered in munc18-1 nulladrenal chromaffin cells

We studied the regulatory effect of Munc18-1 on the cortical F-actin network in neuroen-docrine cells and compared the appearance of the subplasmalemmal network in MCCsfrom wild type and munc18-1 null mice. As shown before (Toonen et al., 2006), theabsence of Munc18-1 in embryonic MCCs resulted in an increased rhodamine-phalloidinlabeling. We used the automated image analysis tool PlasMACC (Kurps et al., 2014)and Chapter 3 of this thesis) to analyze this signal in MCCs from both genotypes. In-stead of measuring the intactness of the cortical network or its number of holes only(Toonen et al., 2006); we combined two aspects (intensity and thickness) of the sig-nal to describe Munc18-1 dependent alterations in the F-actin network (fig4.1A). Wecompared intensity- and thickness profiles of single cells (fig4.1D-E) and quantified thecortical F-actin network in 182 MCCs from seven independent experiments. We foundan increase in the average signal intensity in munc18-1 null MCCs compared to wildtype MCCs (fig4.1B). Besides the enhanced overall signal intensity of 37%, we found athicker actin network (fig4.1C) in MCCs from munc18-1 null mice. The average thick-ness was 21% higher than in the wild type MCCs. These results confirm the publishedF-actin phenotype in MCCs from munc18-1 null mice (Toonen et al., 2006; Kurps andde Wit, 2012; Kurps et al., 2014) and show that both the total actin immunoreactivity(intensity) and the thickness of the network contribute to the altered cortical F-actinnetwork in munc18-1 null MCCs compared to wild type MCCs.

Secretion deficiency per se does not explain F-actin pheno-type in munc18-1 null chromaffin cells

Munc18-1 null MCCs show an almost complete loss of regulated exocytosis (Verhageet al., 2000). The disturbed F-actin regulation might be a contributing factor. To testthis hypothesis, we analyzed the F-actin network in MCCs from snap-25 null and syt-1null mice. Both genotypes display a clear docking and secretion phenotype ((de Witet al., 2009); (Verhage and Sørensen, 2008)). If the disturbed docking mechanism wouldbe the cause of the alteration of the cortical F-actin network in munc18-1 null MCCs,snap-25 null and syt-1 null MCCs are expected to display the same actin phenotype.However, we found no significant difference in the intensity of the F-actin labeling be-tween wild type MCCs and MCCs from Syt-1 deficient (fig4.2A-D) or SNAP-25 deficientMCCs (fig4.2E-H). In MCCs from syt-1 null mice the average F-actin intensity was notsignificantly different from the intensity in wild type MCCs (fig4.2C). The comparisonof the thickness (fig4.2D) of the subplasmalemmal F-actin network also showed no sig-nificant difference. The average F-actin intensity in wild type and snap-25 null MCCswere similar (fig4.2G). The thickness of the cortical network was increased ( 30%) insnap-25 null MCCs (fig4.2H). Hence, the data suggest that a redistribution of actinoccurred in the snap-25 null mutant: while total staining intensity remained the same,the thickness increased. This phenotype is different from the munc18-1 null phenotype,where intensity and thickness of the cortical actin both were increased.

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Figure 4.1: Munc18-1 regulates cortical F-actin A. MCCs labelled with rhodamine-phalloidin to visualize cortical F-actin; left: wild type, right: munc18-1 null, top: red LUT,bottom: grayscale; (B-C) Quantification of rhodamine-phalloidin signal in wild type (dark grey,N:7, n:88) and munc18-1 null (red, N:7, n:94) MCCs. B. intensity: wild type: 16.04 ka.u.±0.66; munc18-1 null: 22.01 ka.u. ± 0.59; t-test, ***, p < 0.001. C. thickness: wild type: 0.25a.u. ± 0.007; munc18-1 null: 0.30 a.u. ± 0.006; t-test, ***, p < 0.001. D-E. Single cell intensityprofiles (D), thickness profiles (E) of wild type and munc18-1 null MCC from panel 2A; shownare mean ± SEM; Scale bars: 2 µm.

Furthermore, these results indicate that the F-actin phenotype in munc18-1 nullMCCs is rather a direct result of the absence of Munc18-1 than the consequence of theimpaired docking and secretion mechanism.

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Figure 4.2: Abolished docking is not sufficient for increase in cortical F-actinnetwork A-B. wild type MCC (A) and syt-1 null MCC (B) labelled with rhodamine-phalloidin.C-D. Quantification of F-actin labelling in wild type (grey, N:1, n:18) and syt-1 null MCCs(green, N:1, n:18); intensity (C) wild type: 17.00 ka.u. ± 1.43; syt-1 null: 19.58 ka.u. ± 1.44;t-test, n.s.; thickness (D) wild type: 0.23 a.u. ± 0.014; syt-1 null: 0.26 a.u. ± 0.015; t-test, n.s.E-F. wild type MCC (E) and snap-25 null MCC (F) labelled with rhodamine-phalloidin. G-H.Quantification of F-actin labelling in wild type (grey, N:1, n:14) and snap-25 null MCCs (lightblue, N:1, n:19); intensity (G) wild type: 14.90 ka.u. ± 2.47; snap-25 null: 14.36 ka.u. ± 1.10;t-test, n.s.; thickness (H) wild type: 0.23 a.u. ± 0.023; snap-25 null: 0.30 a.u. ± 0.010; t-test,**, p=0.009; shown are mean ± SEM; Scale bars: 2 µm.

All heterologous Sec1/Munc18 proteins rescue the F-actinphenotype in munc18-1 null chromaffin cells

The SM protein family and its function in several different intracellular pathways areevolutionary conserved (Toonen and Verhage, 2003). In order to test whether the F-actin regulating role of Munc18-1 is also conserved, we expressed different SM proteinsin munc18-1 null MCCs. We tested the effect of the expression of UNC18, the Sec1orthologue in Caenorhabditis elegans (C. elegans). Munc18-1 and UNC18 are consideredto be involved in the same intracellular pathway and show a relative high amino acidsequence homology (57%, fig4.3A, B). Like Munc18-1 deficient mice, loss of functionmutants of UNC18 show a docking phenotype (Schulze et al., 1994; Weimer et al., 2003).The F-actin regulating function of Munc18-1 might also be shared by UNC18.

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We also expressed mVps33a and mVps45, SM proteins that are known to be im-portant for different intracellular pathways. Vps33a is involved in trafficking from thelate Golgi apparatus to endosomes (Subramanian et al., 2004) and its amino acid se-quence homology with Munc18-1 is 14 % (fig4.3A, B). Vps45 functions in endosomaltrafficking (Bassham et al., 2000; Struthers et al., 2009) (fig4.3A, B) and the homologybetween Munc18-1 and Vps45 is 18 % (fig4.3B). All three proteins, UNC18, mVps33aand mVps45, reduced the subplasmalemmal network in munc18-1 null MCCs. Theaverage intensity in non-treated munc18-1 null MCCs was 27.74 ka.u. ± 1.30. Theexpression of wild type Munc18-1 reduced the intensity by 53% and thereby completelyrescued the F-actin phenotype (fig4.3C). The expression of the UNC18 resulted in sim-ilar reduction of the cortical F-actin (63%). Surprisingly, the expression of the SMproteins mVps33a and mVps45 also strongly decreased the intensity of the cortical F-actin network by 48% and 49%, respectively (fig4.3C; One-way ANOVA; F(4,164)=26.99; p < 0.001). These data show that all SM proteins tested, have a conservedfunction in F-actin regulation.

Most Munc18-1 mutants rescue F-actin and Syntaxin1a phe-notype in munc18-1 null chromaffin cells

In order to pinpoint the F-actin regulating function of Munc18-1 to one or more spe-cific domains or even residues, we tested the expression of several Munc18-1 mutantsin munc18-1 null MCCs. We used lentivirus infection to express mutants, especiallymutations at position Y473 (fig4.4A-C) or S241 (fig4.5D-F). These residues are phos-phorylation sites for different kinases; Y473 for Src kinase and S241 for extracellularsignal-regulated kinase (ERK). Src kinase was recently shown to be involved in actinpolymerization (Olivares et al., 2014). Furthermore, Src kinase regulates the activity ofother actin-regulating proteins (e.g., N-WASP (Banin et al., 1996), cofilin (Kim et al.,2008) and gelsolin (Corte et al., 1997). Therefore, we examined whether the expressionof the phospho-mimicking (Y473D) as well as the non-phophorylatable mutant (Y473A)in munc18-1 null MCCs has an effect on the increased cortical F-actin network (fig4.4A-C). Both mutants reduced the intensity of the fluorescent signal to the same extent asthe expression of wild type Munc18-1. Besides the increase in cortical F-actin, munc18-1 null MCCs also show a strongly reduced amount ( 50%) of Syntaxin1a (Voets et al.,2001). Therefore, we analyzed the effect of the expression of Munc18-1 mutants on theSyntaxin1a level in munc18-1 null MCCs (fig4.4C). The intensity of Syntaxin1a at thePM was comparable after the expression of wild type Munc18-1 and both mutants.

Phosphorylation by ERK is a known mechanism to regulate actin reorganization(Chu et al., 2000). The expression of the phospho-mimicking (S241D) as well as thenon-phophorylatable mutant (S241A) resulted in F-actin intensity values below wildtype Munc18-1 (Munc18 vs Munc18S241A; *; Munc18 vs Munc18S241D; *). To excludeexpression level dependent effects, we quantified the cellular levels of Munc18-1 usingimmunocytochemistry. No significant differences were observed (fig4.5F). Therefore weconcluded that the S241 mutations potentiate Munc18-1s ability to reduce cortical F-actin, but the phosphorylation status of this residue does not influence this aspect ofMunc18-1 function.

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Figure 4.3: SM proteins rescue the F-actin phenotype in munc18-1 null MCCsA. Schematic overview of involvement of SM proteins in distinct intracellular pathways (Nu:Nucleus, ER: Endoplasmatic reticulum, GA: Golgi apparatus, En: Endosome, Vac/Lys: Vac-uoles/Lysosomes). B. amino acid sequence homology between used SM proteins, resulting frompairwise alignment algorithm by Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo).C. munc18-1 null MCCs, either native or expressing different SM proteins, top row: rhodamine-phalloidin labeling, middle row: eGFP signal due to SFV virus expression, bottom row: merge ofboth signals. D. Quantification of cortical F-actin intensity at the PM in non-treated munc18-1null MCCs (red, N:5, n:68; 27.74 ka.u. ± 1.30) and munc18-1 null MCCs expressing Munc18-1(grey, N:5, n:45; 12.94 ka.u. ± 1.04), UNC18 (dark green, N:3, n:26; 10.13 ka.u. ± 1.49), Vps33a(orange, N:4, 16; 14.48 ka.u. ± 2.08) and Vps45 (light blue, N:2, n:18; 14.07 ka.u. ± 1.74);One-way ANOVA; F(4,164)= 26.99; p < 0.001 (post hoc t-test with Bonferroni correction: forall SM proteins compared to munc18-1 null (***, p < 0.0001); for all comparisons between SMproteins (n.s.)); shown are mean ± SEM; Scale bars: 2 µm.

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Figure 4.4: Phosphorylation-locked Munc18-1 mutants rescue F-actin phenotypeA. Confocal images of munc18-1 null MCCs, expressing wild type Munc18-1 or the mutantvariants Munc18Y473A and Munc18Y473D, top row: rhodamine phalloidin signal to visual-ize cortical F-actin, second row: Syntaxin1a antibody staining, third row: eGFP signal fromCre-virus, bottom row: merge of all three channels. B. Quantification of cortical F-actin in-tensity in munc18-1 null MCCs, expressing wild type Munc18-1 (N:1, n:7; 19.66 ka.u. ± 1.44),Munc18Y473A (N:1, n:6; 24.71 ka.u. ± 4.10), Munc18Y473D (N:1, n:8; 17.64 ka.u.± 1.69);One-way ANOVA; F(2,18) = 2.11; p=0.15; n.s. C. Quantification of Syntaxin1a staining inten-sity at the PM in munc18-1 null MCCs, expressing wild type Munc18-1 (N:1, n:7; 20.02 ka.u.± 3.74), Munc18Y473A (N:1, n:6; 23.32 ka.u. ± 3.45), Munc18Y473D (N:1, n:8; 28.56 ka.u. ±6.24); One-way ANOVA; F(2,18) = 2.20; p=0.14; n.s.; shown are mean ± SEM; Scale bars: 2µm.

Munc18V236T rescues the Syntaxin1a phenotype, but notthe F-actin phenotype

We subsequently examined the F-actin-regulating ability of a group of Munc18-1 mu-tants that contained point mutations at residues that differ between Munc18-1 and itsisoform Munc18-2, but are otherwise highly conserved in Sec1 orthologues. An overviewof typical examples of the three different signals in all conditions is shown in fig4.6A.Quantification of the cortical F-actin signal intensities clearly showed that expressionof most Munc18-1 mutants resulted in decreased cortical F-actin, similar to that of wildtype Munc18-1. Surprisingly, one Munc18-1 mutant (Munc18V263T) did not rescuethe increased cortical F-actin network (fig4.6A). Besides the cortical F-actin phenotype,munc18-1 null MCCs show reduced levels (≈ 50% reduction) of Syntaxin1a (Voets et al.,2001). Syntaxin1a intensities in all mutant samples were highly similar to the cells ex-pressing wild type Munc18-1. Only the expression of Munc18-1 mutant Munc18D17Gresulted in a significant increase in Syntaxin1a intensity at the PM. These data showthat the expression of Munc18V263T in munc18-1 null MCCs rescues the Syntaxin1aphenotype, but not the cortical F-actin phenotype. We repeated the experiment andtested Munc18V263T levels and cellular distribution (fig4.8A). The quantification ofthe cortical F-actin levels confirmed the findings of the previous experiment (fig4.8B).

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Figure 4.5: Phosphorylation-locked Munc18-1 mutants rescue F-actin phenotypeD. Confocal images of munc18-1 null MCCs, expressing wild type Munc18-1 or the mutantvariants Munc18S241A and Munc18S241D, top row: rhodamine phalloidin signal, middle row:Munc18-1 antibody staining, bottom row: merge of both channels. E. Quantification of corticalF-actin intensity in munc18-1 null MCCs, expressing wild type Munc18-1 (N:1, n:13; 21.00 ka.u.± 1.49), Munc18S241A (N:1, n:14; 16.26 ka.u. ± 0.89), Munc18S241D (N:1, n:13; 15.01 ka.u. ±1.19); One-way ANOVA; F(2,37) = 6.64; p=0.003; post hoc t-tests with Bonferroni correction:Munc18 vs Munc18S241A;*, p=0.010; Munc18 vs Munc18S241D;*, p=0.005; Munc18S241Avs Munc18S241D; n.s. F. Quantification of Munc18-1staining intensity in the whole cell inmunc18-1 null MCCs, expressing either wild type Munc18-1 (N:1, n:13; 502.93 a.u. ± 87.01),Munc18S241A (N:1, n:14; 488.03 a.u. ± 80.70), Munc18S241D (N:1, n:13; 577.16 a.u. ±120.61); One-way ANOVA; F(2,37) = 0.24; p=0.79; shown are mean ± SEM; Scale bars: 2 µm.

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Figure 4.6: Munc18V236T can not rescue the F-actin phenotype A. Confocal imagesof munc18-1 null MCCs infected with either wild type Munc18-1 (N:1, n:6) or six Munc18-1 mu-tants with point mutations (Munc18D17G: N:1, n:8; Munc18R171A: N:1, n:9, Munc18V263T,N:1, n:11; Munc18K25D: N:1, n:7, Munc18R505P: N:1, n:11, Munc18A226T: N:1, n:8). Scalebars: 2 µm.

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Figure 4.7: Munc18V236T can not rescue the F-actin phenotype B. Quantificationof cortical F-actin signal intensity: wild type Munc18-1: 21.07 ka.u. ± 2.75; Munc18D17G:23.14 ka.u. ± 2.54; Munc18R171A: 17.01 ka.u. ± 2.10; Munc18V263T: 39.22 ka.u. ± 4.38;Munc18K25D: 21.29 ka.u. ± 2.95; Munc18R505P: 22.26 ka.u. ± 2.09; Munc18A226T: 16.88ka.u. ± 2.67; One-way ANOVA: F(6,53) = 7.50; p < 0.001; post hoc t-tests with Bonferronicorrection indicated three statistically significant differences (Munc18V263T vs Munc18R171A:*, p=0.0004; Munc18V263T vs Munc18R505P: *, p=0.0023; Munc18V263T vs Munc18A226T:*, p=0.0008). C. Quantification of Syntaxin1a signal intensity at the PM: wild type Munc18-1: 15.98 ka.u. ± 1.54; Munc18D17G: 24.30 ka.u. ± 2.36; Munc18R171A: 18.95 ka.u. ±2.47; Munc18V263T: 19.45 ka.u. ± 2.63; Munc18K25D: 18.27 ka.u. ± 1.57; Munc18R505P:15.57 ka.u. ± 1.20; Munc18A226T: 14.11 ka.u. ± 1.67; One-way ANOVA: F(6,53) = 2.39;p=0.04; post hoc t-tests with Bonferroni correction showed only significant difference betweenthe expression of Munc18D17G and Munc18R505P; shown are mean ± SEM.

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Figure 4.8: Level and localization of Munc18V263T are similar to wild typeMunc18-1 A. munc18-1 null MCCs, infected with lentivirus particles that express either eGFP(top row), wild type Munc18-1 (middle row) or Munc18V263T (bottom row); shown are F-actinstainings (1st column), eGFP signals (2nd column), Munc18-1 stainings (3rd column) and themerged images from the three channels ( 4th column). B. Quantification of cortical F-actin in-tensity: eGFP: N:1, n: 15; 18.43 ka.u. ± 3.70; wild type Munc18-1: N:1, n:12; 7.94 ka.u. ± 2.88;Munc18V263T: N:1, n:14; 21.16 ka.u. ± 4.07; One-way ANOVA: F(2,38) = 21,78; p < 0.001;post hoc t-tests with Bonferroni correction: eGFP vs Munc18: ***, p < 0.0003; Munc18V263Tvs Munc18: ***, p < 0.0003; eGFP vs Munc18V263T: n.s. C. Analysis of Munc18-1 stainingintensity in the entire cell: eGFP: N:1, n: 15; 42.23 a.u. ± 11.73; wild type Munc18-1: N:1,n:12; 493.49 a.u. ± 88.85; Munc18V263T: N:1, n:14; 712.91 a.u. ± 116.68; One-way ANOVA:F(2,38) = 6.64; p = 0.004; post hoc t-tests with Bonferroni correction: eGFP vs Munc18: *,p=0.006; Munc18V263T vs Munc18: n.s.; eGFP vs Munc18V263T: *, p= 0.0036; shown areaverages ± SEM; Scale bars: 2 µm.

The intensity levels of the Munc18-1 staining were not significantly different in MCCsthat expressed either wild type Munc18-1 (≈ 12 fold increase over background) or theV263T mutant (≈ 17 fold increase over background) (fig4.8C). These data show thatexpression and localization of Munc18V263T are similar to wild type Munc18-1.

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Summary of all tested Munc18-1 mutants

To summarize and compare the effect sizes of the different Munc18-1 mutants on thecortical F-actin network, the signal intensities from several experiments were normalizedto the signal intensity of the wild type Munc18-1 condition (fig4.9A). The Munc18-1mutants were arranged based on the localization of the mutated residue. Both mutantsin domain 1 (Munc18D17G and Munc18K25D) were similar to wild type Munc18-1.All point mutations in domain 2a (R171A, A226T, S241A, S241D) showed a slightdecrease in the F-actin intensity values compared to wild type levels. Mutations ofresidues in domain 3 (V263T, Y473A, Y473D) showed the biggest effect size. EspeciallyMunc18V263T stood out with an 86 % increase compared to wild type Munc18-1. Thesignal intensity in the MCCs expressing the Munc18 mutant with the mutation indomain 2b (Munc18R505P) did not differ significantly from signal intensities in thewild type condition.

In conclusion, mutants with alterations in domain 2a consistently showed an in-creased ability to decrease the intensity of the cortical F-actin to values even lower thanin the wild type situation, whereas mutations in domain 3 resulted in a decreased abil-ity to regulation the cortical F-actin. To compare the effect of the Munc18-1 mutantson the Syntaxin1a mistargeting phenotype, we analyzed the ratio of PM-localized andcytoplasmic Syntaxin1a for several Munc18-1 mutants. In munc18-1 null MCCs thisratio is lower than in wild type MCCs (normalized to 1), where Syntaxin1a is primarilylocalized at the PM (Kurps et al., 2014). All tested mutants show a ratio similar towild type levels. Therefore, we conclude that cortical F-actin regulation and Syntaxin1achaperoning are two independent aspects of Munc18-1 function.

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Discussion

In the present study we describe Munc18-1 dependent cortical F-actin regulation inadrenal MCCs. We characterize two different aspects (intensity and thickness) of theincreased cortical F-actin network inmunc18-1 null MCCs and show that this phenotypeis Munc18-1 specific and not caused by impaired secretion in general. Based on theexpression of Munc18-1 orthologues and Munc18-1 mutants in munc18-1 null MCCs,we show that several other SM proteins and most Munc18-1 mutants share the F-actin regulating function of Munc18-1. We identify one Munc18-1 mutant with a pointmutation in domain 3 that results in an impaired rescue ability.

F-actin regulation is a conserved feature of all SM proteinsAll SM proteins studied here, regulate cortical F-actin in mammalian CCs: SM proteinsthat are involved in regulated exocytosis (like Munc18-1) but in a different species(e.g., UNC18 in C. elegans) or involved in different membrane trafficking pathways,within the same species (e.g., mVps33a, mVps45). The general mechanisms regulatingactin polymerization appear to be similar among different species. For example, Cdc42is essential for actin polarization in S. cerevisiea (Adams et al., 1990; Adamo et al.,2001), in C. elegans (Chen et al., 1993) and in mammals (Aepfelbacher et al., 1994).Similarly, the actin-regulating role of the Arp2/3 complex was discovered in S. cerevisiea(Moreau et al., 1996) and later shown in C. elegans (Shakir et al., 2008) and in mammals(Machesky and Insall, 1998).

Sec1, the ancestral orthologue of Munc18-1 in S. cerevisiea, was suggested to in-teract directly with the actin cytoskeleton (Egerton et al., 1993) and Sec1 mutantsresulted in a depolarized actin cytoskeleton (Mulholland et al., 1997). More recentstudies showed that actin dysregulation is observed in several yeast mutants of the latesecretory pathway, including sec1 (Aronov and Gerst, 2004). The expression of UNC18,the Munc18-1 orthologue with the highest amino acid sequence homology, showed anactin regulating ability comparable to Munc18-1. Previously, the conserved function ofUNC18 and Munc18-1 in the docking mechanism was described (Weimer et al., 2003;Gracheva et al., 2010). Our results suggest a similar conservation of the actin-regulatingfunction of both proteins.

Surprisingly, the expression of endosomal SM proteins (Vps33a, Vps45) in munc18-1 null CCs also led to a reduction of the cortical F-actin network. To our knowledge,no direct links between Vps33a or Vps45 and actin are described. However, actinfilaments are also important for endocytosis and for vesicle trafficking processes betweenearly and late endosomes or endosomes and vacuoles (for review see (Kaksonen et al.,2006). Therefore it is likely that endosomal SM proteins are also able to regulate actin(Richardson et al., 2004). Vps33a is part of the HOPS complex (Seals et al., 2000) andthe inhibition of the HOPS complex in S. cerevisiea results in blocked actin enrichmentat the vertex ring which is essential for homotypic vacuole fusion (Karunakaran et al.,2012). Expression of Vps33a using SFV particles results in an overload of Vps33a in theCCs. This in turn might have led to an increase in non-functioning or inhibited HOPScomplexes, which block actin enrichment. Another study showed that the expressionof Vps18, a component of the HOPS complex like Vps33, resulted in the recruitmentof other HOPS complex components, actin and actin-regulating proteins (e.g., ezrin)to lysosomes and vacuoles (Poupon et al., 2003). Therefore, the expression of Vps33might give rise to the recruitment of actin regulating proteins that reverse the increasein the cortical F-actin network in munc18-1 null CCs.

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Domain1 Domain2bDomain3Domain2a

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Figure 4.9: Overview of all tested Munc18-1 mutants A. Quantification of corticalF-actin intensities (normalized to wild type Munc18-1) in munc18-1 null MCCs, expressingeither wild type Munc18-1 (N:4, n:38), eGFP (N:2, n:27; 191.43 a.u. ± 16.16) or Munc18-1mutants: Munc18D17G (N:1, n:8; 109.82 a.u. ± 12.06), Munc18K25D (N:1, n:7; 101.03 a.u.± 13.99), Munc18R171A (N:1, n:9; 80.72 a.u. ± 9.98), Munc18A226T (N:1, n:8; 80.14 a.u. ±10.77), Munc18S241A (N:1, n:14; 77.44 a.u. ± 4.25), Munc18S241D (N:1, n:13; 71.83 a.u. ±5.68), Munc18V263T (N:2, n:25; 230.22 a.u. ± 17.16), Munc18Y473A (N:1, n: 6; 125.67 a.u.± 20.87), Munc18Y473D (N:1, n:8; 89.74 a.u. ± 8.58) Munc18R505P (N:1, n:11;105.63a.u.±9.92); One-way ANOVA: F(11,162) = 17.45; p < 0.001; post hoc t-tests with Bonferroni cor-rection showed significant differences for wild type Munc18-1 vs eGFP: ***, p < 0.0000152;wildtype Munc18-1 vs Munc18V263T: ***, p < 0.0000152. B. Quantification of ratio (cytoplasmicintensity/PM intensity) of Syntaxin1a signal (normalized to wild type Munc18-1) in munc18-1null MCCs, expressing either wild type Munc18-1 (N:3, n:33), no Munc18-1 (N:1, n:17; 0.66± 0.035) or Munc18-1 mutants: Munc18D17G (N:1, n:9; 1.16 ± 0.0079), Munc18K25D (N:1,n:9; 1.24 ± 0.066), Munc18R171A (N:1, n:11; 1.07 ± 0.120), Munc18A226T (N:1, n:8; 1.12± 0.141), Munc18V263T (N:1, n:13; 1.24 ± 0.108), Munc18Y473A (N:1, n: 8; 0.90 ± 0.100),Munc18Y473D (N:1, n:9; 0.92 ± 0.0067) Munc18R505P (N:1, n:9;1.24 ± 0.141); One-wayANOVA: F(9,111) = 5.43; p < 0.001; post hoc t-tests with Bonferroni correction showed signif-icant difference for wild type Munc18-1 vs munc18-1 null: ***, p < 0.000022; shown are mean± SEM.

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Furthermore, despite a low overall sequence similarity, the 3D structure of endoso-mal SM proteins is expected to be at least similar to the exocytotic orthologues, giventhe fact that they bind the N-terminals of Syntaxins (Bracher and Weissenhorn, 2002;Khvotchev et al., 2007). Our data shows that this functional similarity extends to theregulation of actin in subplasmalemmal regions.

Munc18-1 mutants and specific domainsThe largest differences in cortical F-actin levels when compared to the expression of wildtype Munc18-1 were observed for mutants R171, A226, S241 and V263. The first threeshowed a slightly higher ability to rescue the cortical F-actin phenotype of munc18-1null CCs, whereas the last mutant was completely unable to rescue the phenotype. Wesubsequently assigned all tested mutants to their specific domains and showed that all”gain-of-function” mutants were mapped to domain 2a, whereas all mutants in domain3 led to a decreased ability to regulate cortical F-actin and are therefore classified as”loss-of-function” mutations. These findings implicate that domain 3 plays an essentialrole in the Munc18-1 dependent cortical F-actin regulation. The Syntaxin1a distribu-tion was rescued by all tested mutants. The mutants mapped to domain 1, 2a and2b showed a higher ratio of membrane/cytoplasm Syntaxin1a levels compared to theexpression of wild type Munc18-1. Therefore those mutants are ”gain-of-function” mu-tants. In contrast to the effect of domain 3 mutants on the cortical F-actin, their effecton the Syntaxin1a distribution is not uniform. The expression of the V263 mutantresulted in an increased membrane localization, whereas both Y473 mutants showeddecreased Syntaixn1a levels on the PM. These data implicate that domain 3 mutantsof Munc18-1 have bi-directional effects on the distribution of Syntaxin1a. Domain 3ais crucial for Munc18s role in the priming step of exocytosis (Han et al., 2013) and theloop in domain 3a is essential for regulated exocytosis (Martin et al., 2013), but not forchaperoning of Syntaxin1a to the PM. Since the regulation of subplasmalemmal F-actinis functionally linked to regulated exocytosis in chromaffin cells, these findings confirmthe importance of domain 3 in Munc18-1.

Importance of hydrophobicityResidue V263, which appears to be crucial for the actin regulating function of Munc18-1, is evolutionary conserved in its orthologues Rop-1 (Drosophila melanogaster) andUNC18 (C.elegans). However, alignment studies showed an Isoleucine (I) residue inSec1 (S.cerevisiea), an alanine (A) residue in Vps33 and again an Isoleucine (I) residuein Vps45 at the corresponding position. Isoleucine and alanine are structurally similarto valine and all three amino acids contain hydrophobic side chains, suggesting that thisproperty is relevant at this position for the role of SM-proteins in regulation of corticalF-actin. Hydrophobicity at this position may be important for potential interactionswith lipid membranes.

The pathway hypothesis: an update and a to-do listSince there is no clear evidence that Munc18-1 can directly interact with actin monomersor filaments, an indirect regulatory mechanism is more likely. The importance of hy-drophobic residues for Munc18-1 dependent cortical F-actin regulation, hints at a po-tential involvement of lipids. Our current working model assumes a prominent role ofthe phospholipid PI(4,5)P2 as a link between Munc18-1 and the F-actin cortex. Thishypothesis is supported by three lines of evidence: (I) Munc18-1 was shown to inhibitphospholipase D (PLD) by direct interaction (Lee et al., 2004) and this finding wasconfirmed by yeast-3-hybrid studies (Angeli Moller, MDC, Berlin, unpublished data).

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(II) PLD can be linked to PI(4,5)P2 generation by a specific kinase (Jenkins et al.,1994; Exton, 1997). (III) The phospholipid PI(4,5)P2 has a multitude of intracellularfunctions, including membrane anchoring of the actin cytoskeleton and actin regulat-ing proteins (for review see: (McLaughlin et al., 2002; Logan and Mandato, 2006).In the following chapter we will test this hypothesis by analyzing potential Munc18-1dependent alterations in PI(4,5)P2 levels.

Material and Methods

Laboratory animalsGeneration of munc18-1 null mice was described earlier (Verhage et al., 2000). Embryos(E18) were obtained by caesarean section of pregnant females from timed breeding ofheterozygotes. Laboratory animals were bred and housed according to Dutch govern-mental guidelines for animal welfare.

Chromaffin cell primary cultureAdrenal chromaffin cells from embryonic wild type, munc18-1 null, snap-25 null andsyt-1 null (E18) were isolated as described previously (Sørensen et al., 2003b). Theisolated chromaffin cells (E18) were cultured on glass coverslips coated with rat tailcollagen.

Semliki Forest virus (SFV) and Lentivirus (LV) infectionBefore the virus particles were added to the culture medium, the viruses were activatedwith chymotrypsin and aprotinin. MCCs were infected with 25 µl of SFV containingsolution per well and incubated for 6-9 hrs at 37 ◦C. Lentivirus: 8 µl of lentivirus so-lution was added to the medium in each well to infect MCCs with lentiviruses. TheMCCs were incubated for 3048 hrs at 37 ◦C.

ImmunocytochemistryChromaffin cells were fixed with 4% paraformaldehyde (PFA) at DIV3. The cells werepermeabilized by 5 min incubation in phosphate buffered saline (PBS) containing 0.5%Triton X-100. To block nonspecific binding, the cells were incubated for 30 min in PBScontaining 0.1% Triton X-100 and 2% normal goat serum. All antibodies were diluted inthis solution. The cells were incubated in the primary antibody solution for 1 h at roomtemperature (RT). After washing 3 times with PBS for 10 min, the cells were incubatedin the secondary antibody solution for 1 hr at RT. The cells were washed 3 times inPBS and mounted on microscopy slides with Mowiol in preparation for the confocalmicroscopy and glycerol for the structured illumination microscopy, respectively.

AntibodiesSpecific primary antibodies were used against Syntaxin1a (rabbit polyclonal, I379 (giftfrom Sudhof lab), 1:1000) and Munc18-1. As secondary antibodies goat α-mouse andgoat α-rabbit Alexa Fluor 488 and 647 were used (all Molecular Probes, 1:1000).TheF-actin network was stained with the conjugate rhodamine-phalloidin (R415, MolecularProbes), which does not require a secondary antibody.

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Image AcquisitionImages of fixed and mounted samples were acquired using confocal microscopy andstructured illumination microscopy. For confocal microscopy, the microscopy slides wereimaged with a 63x plan-neofluor lens (numerical aperture 1.4, Carl Zeiss) on a Zeiss 510Meta Confocal Microscope. An additional zoom factor of 5 was applied and the imageswere acquired with a frame size of 1024x1024 pixels. A single image of the equatorialplane of the MCCs was acquired. Structured illumination microscopy was performedwith the Elyra PS.1 platform (Carl Zeiss), equipped with a 63x plan-apochromat lens(numerical aperture 1.46, Carl Zeiss). Here a z -stack covering the entire cell was ac-quired.

Image AnalysisThe analysis of the images was primarily executed with PlasMACC, which is imple-mented as a plugin in the image analysis software ImageJ. We determined intensity,thickness and density of fluorescent signals at the PM and in subplasmalemmal regions.We performed Students t-tests to compare the described parameters between wild typeand munc18-1 null CCs. When we compared more than two groups, we first performedan One-way ANOVA to determine the overall statistical significance between groups. Ifwe found significant differences, we performed post hoc t-tests with Bonferroni correc-tions to specify which groups differed significantly.

Clustal Omega alignment quantificationAmino acid sequence homology between SM proteins was generated and calculatedwith the ClustalW2 algorithm (http://www.ebi.ac.uk/Tools/msa/clustalw2/), which isincluded in the services on the EMBL-EBI homepage. The input files from all SM pro-teins were downloaded from the Uniprot website in FASTA format.

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