1. Volume 30, Issue 6, Pages 746-758 (September 2014)
Crosstalk between PI(4,5)P2 and CK2 Modulates Actin Polymerization during Endocytic Uptake 
Isabel María Fernández-Golbano, Fatima-Zahra Idrissi, Jonathan P. Giblin, Bianka L. Grosshans, Virginia Robles, Helga Grötsch, María del Mar Borrás, María Isabel Geli 
Developmental Cell 
Volume 30, Issue 6, Pages (September 2014)
DOI: /j.devcel
Copyright © 2014 Elsevier Inc. Terms and Conditions

2. Figure 1
Phosphorylation of Myo5 S1205 Downregulates the Myo5/Vrp1 Interaction and Myo5-Induced Actin Polymerization
(A and B) Autoradiography (Autorad.) of a Coomassie stained SDS-PAGE gel (Coom.) of the depicted GST-Myo5 fusion proteins (1–4; A) or GST-Myo5-Cext WT (MYO5) or S1205A or S1205E mutants (B) incubated with a yeast extract and γ-32P-ATP.
(C) Fluorescence micrographs (left panel) of beads coated with GST-Myo5-Cext WT (MYO5) or S1205C or S1205D mutants, incubated with yeast extracts and rhodamine-actin. The graph indicates the normalized average actin patch density, the SEM, and Student’s t test p values (n > 30, from at least three independent experiments).
(D) Anti-MYC (Arp3-MYC) and anti-HA (Vrp1-HA) immunoblots of pull-downs performed with beads coated with GST-Myo5-Cext WT (MYO5) or S1205C or S1205D mutants, from extracts of yeast expressing either Arp3-MYC (n = 3) or Vrp1-HA (n = 5). Ponceau red was used to detect the GST-fusion proteins. The graph indicates the Av, SEM, and Student’s t test p values of the Arp3-MYC and Vrp1-HA signals for the S1205C and S1205D mutants normalized to the WT.
(E) Graphs representing the Av, SEM, and Student’s t test p values of the β-galactosidase activity of yeast bearing the reporter under the control of the LexA operon, cotransformed with plasmids encoding the LexA DNA binding domain fused to the Myo5-Cext WT or the S1205C or S1205D mutants and the B42 transcriptional activator fused to ARC40 or VRP1. The S1205C and S1205D values are normalized to the WT (n = 5).
(F) PAP and anti-HA immunoblots of IgG-Sepharose pull-downs from cells expressing Vrp1-HA and Protein A (PA)-tagged WT or mutant Myo5.
The graph indicates the Av, SEM, and Student’s t test p values of the Vrp1-HA signal coprecipitating with the S1205D and S1205C Myo5 mutants, normalized to the WT Myo5 (n = 3). See also Figure S1.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 2
Cka2 Phosphorylates Myo5 S1205 and Downmodulates Myo5-Induced Actin Polymerization
(A and B) Autoradiography (Autorad.) of Coomassie stained SDS-PAGE gels (Coom.) showing GST-Myo5-Cext or GST-Myo5-S1205C-Cext, incubated with γ-33P-ATP and extracts from strains bearing the indicated mutations or the isogenic WTs (SCMIG1182 pCKA2-leuΔ::URA3, left; and BY4741, right; A) or extracts from either WT (BY4741) or ckb1Δ ckb2Δ cells overexpressing the indicated CK2 subunits or bearing the empty plasmid (empty; B).
(C) Anti-HA immunoblot showing expression or overexpression (+) of the indicated HA-tagged CK2 catalytic subunits.
(D and E) Fluorescence micrographs (upper panel) of beads coated with GST-Myo5-Cext WT (MYO5) or S1205D or S1205C mutants, incubated with rhodamine-actin and yeast extracts from either WT, cka1Δ, or cka2Δ strains (D) or extracts from WT cells bearing a multicopy plasmid either empty, or encoding the indicated CK2 subunits (E).
The graphs indicate the Av, SEM, and Student’s t test p values of the normalized patch density (n > 30, from three experiments). See also Figure S2 and Table S1.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

4. Figure 3
Constitutive Phosphorylation of Myo5 S1205 by Cka2 Interferes with Progression of the Actin-Dependent Phase of Endocytosis
Fluorescence micrographs of 2 s consecutive frames from two-color time-lapse movies of myo3Δ myo5Δ ABP1-mRFP (A) or myo3Δ myo5Δ SLA1-mCherry cells (B), expressing WT GFP-Myo5 or the S1205C or S1205D mutants and either bearing an empty plasmid or a multicopy vector for overexpression of the indicated CK2 subunits (+CKA1, +CKA2, +cka2-K79A). The Av ± SEM of the corresponding lifespan (left; A) or the arrival time of Myo5 versus Sla1 (arrows; B) and the Student’s t test p values are indicated (n = 75). Only long-lived (lifespan > 16 s) GFP-Myo5-S1205D or GFP-Myo5 +CKA2 patches were analyzed in (B). Graphs in (A) represent the frequency distribution of the lifespan of GFP-Myo5 and Abp1-mRFP in the myo3Δ GFP-MYO5 ABP1-RFP (WT) myo3Δ GFP-myo5-S1205D ABP1-RFP (S1205D) or myo3Δ GFP-MYO5 ABP1-RFP +CKA2 (+CKA2) strains.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

5. Figure 4
Phosphorylation of Myo5 S1205 by CK2 Is Required to Constrict the Neck of Endocytic Invaginations
(A) Fluorescence micrographs of 2 s consecutive frames from two-color time-lapse movies of either myo5Δ, myo5Δ cmd1-226 or myo5Δ cka2Δ cka1-13 strains coexpressing Abp1-mCherry and GFP-Myo5 WT or the S1205C mutant. The Av ± SEM of the corresponding lifespan (left), the departure time (arrow) of Myo5 with respect to Abp1, and the Student’s t test p values are indicated (n = 75). The graphs represent the frequency distribution of the lifespan of GFP-Myo5 and Abp1-mCherry in indicated strains.
(B) Electron micrographs of PM invaginations labeled with immunogolds against HA in WT or cka2Δ cka1-13 cells expressing the indicated WT and mutant Myo5-HA, and scheme of the geometrical parameters describing the morphology of the invaginations and the position of the gold particles. IL, invagination length; ID, invagination neck diameter. ID for unconstricted profiles corresponds to the diameter of the tubule at 40 nm from the invagination tip; for invaginations with a constricted neck, the diameter was measured at the constriction. GDPM, gold distance to the PM; GDLB, gold distance to the lipid bilayer; GRP, gold relative position. GRP, GDPM/IL. Graphs below represent the Av ± SEM of the invagination lengths or neck diameters for the indicated strains. The diameters for the populations of invaginations with 50 nm < IL < 100 nm or > 100 nm are shown, when indicated. The Student’s t test p values and the population size (N) are indicated.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

6. Figure 5 Cka2 Is Associated with the Endocytic Machinery at the PM
(A) Autoradiography (Autorad.) of a Coomassie stained SDS-PAGE gel (Coom.) showing GST-Myo5-Cext or GST-Myo5-S1205C-Cext, incubated with γ-33P-ATP and WT yeast fractions enriched in PM (P13) and cytosol (S100; left) or cytosols (S100) from WT or ckb1Δ ckb2Δ cells, as indicated (right).
(B) Immunoblots against the indicated proteins of equivalents of yeast P13 and S100 fractions used in A, demonstrating the amounts of Cka2-HA and the partition of the PM marker Gas1 (PM) and the cytosolic marker Hxk1 (Cyt.). The equivalent loading was 1 and 15 μg for the P13 and the S100 fractions, respectively.
(C) Anti-HA immunoblots of native PAGE gels of yeast fractions enriched in cytosol (S100) and PM (P13) from a WT or a ckb1Δ ckb2Δ strain expressing CKA2-HA, solubilized with digitonin or SDS.
(D) Immunoblots against the indicated proteins of total (T), PM, cytosolic, or microsomal (Mic.) fractions prepared from WT cells expressing CKA2-HA and CKA1-MYC. Gas1, Hxk1, and Pep12 are PM (PM), cytosolic (Cyt.) and trans-Golgi network-endosomal (TGN and End.) markers, respectively. Ten microgram of protein was loaded per lane.
(E) Immunoblots against the indicated proteins of anti-HA or anti-MYC antibody-conjugated Sepharose pull-downs from PM fractions of WT cells expressing CKA2-HA and CKA1-MYC or the isogenic untagged strain (CKA1 CKA2).
(F) Immunoblot against Cka2-HA of glutathione Sepharose beads coated with the indicated GST fusion proteins purified from E. coli and incubated with Cka2-HA purified from yeast.
See also Figure S3.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

7. Figure 6
CK2 and the PI(4,5)P2-5 Phosphatases Sjl1 and Sjl2 Share Endocytic Functions
(A) Serial dilutions of sjl1Δ sjl2Δ cells or the isogenic WT overexpressing the indicated CK2 catalytic subunits or bearing an empty plasmid (left) or overexpressing the indicated WT or mutant Myo5 (right).
(B) Fluorescence micrographs of sjl1Δ sjl2Δ cells or the isogenic WT overexpressing the indicated CK2 catalytic subunits or bearing an empty plasmid, incubated for 1 hr with Lucifer yellow. Scale bar represents 2 μm. Graph represents the Av ± SEM and the Student’s t test p values of the percentage of cells with vacuolar Lucifer yellow (eight different fields with more than 25 cells).
(C) PAP, anti-Cmd1, and anti-HA immunoblots of IgG-sepharose pull-downs from WT or sjl1Δ sjl2Δ cells expressing PA-Myo5 and Vrp1-HA as indicated.
(D) Electron micrographs of PM invaginations labeled with immunogolds against actin in WT and sjl1Δ sjl2Δ cells. Graphs represent the Av ± SEM of the invagination lengths (IL; left) or neck diameters (right) for invaginations of the indicated strains, and for the invaginations with 50 nm < IL < 100 nm or > 100 nm, when indicated.
(E) Electron micrographs of PM invaginations labeled with immunogolds against HA of cells expressing Slj2-HA. Graphs represent the frequency of gold particles against Slj2-HA decorating the base and neck (GRP < 0.7) or the tip (GRP > 0.7) of invaginations shorter than 100 nm, and the Av ± SEM and the Student’s t test p values of the gold distance to the lipid bilayer (GDLB; right) for immunogolds labeling Sjl2-HA, Sla2-HA, Sla1-HA, or Myo5-HA with GRP < 0.5 or 0.5 ≤ GRP < 1 (n = 120 for each protein).
See Figure 4B for details of the parameters used. Information on the GDLB for Sla1-HA, Sla2-HA, and Myo5-HA are extracted from Idrissi et al., 2008, See also Figure S4.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

8. Figure 7
The Synaptojanin Substrate PI(4,5)P2 Releases Autoinhibition of Myo5 and Inhibits Cka2
(A) Phosphoinositide lipid strips overlaid with equimolar amounts of the purified Myo5 Cext (PA-Cext) or neck and TH1 (PA-nT) domains fused to PA, detected using PAP.
(B) PAP (PA-Myo5), anti-Cmd1, or anti-HA (Vrp1-HA) immunoblots of IgG-Sepharose pull-downs from cells expressing Vrp1-HA and PA-Myo5 in the presence of artificial liposomes containing either phosphatidyl-choline (PC) alone, or combined with 50% phosphatidyl serine (PS), or 25% PS and 5% PI(4,5)P2. Graphs show the Av ± SEM and Student’s t test p values of the signals of Cmd1 and Vrp1, normalized to the PC sample.
(C) PAP and anti-Cmd1-immunoblots of IgG-Sepharose pull-downs from myo5Δ cells expressing the PA-Myo5 constructs lacking the Cext (HnT) or the TH1 domain (HnCext) in the presence of artificial liposomes containing PC combined with either 50% PS or 25% PS and 5% PI(4,5)P2.
(D) Phosphoinositide lipid strips overlaid with purified GST or GST-Cka2 detected with an anti-GST antibody.
(E) Native gel showing the release of monomeric Cka2 from the cytosolic holoenzyme in the presence of increasing concentrations of PI(4,5)P2 diC8. The graph represents the band intensity of monomeric Cka2 normalized to the sample without PI(4,5)P2.
(F) Autoradiography (Autorad.) of a Coomassie stained SDS-PAGE gel (Coom.) showing GST-Myo5-Cext of the WT (MYO5) or the S1205C mutant, incubated with γ-33P-ATP and purified Cka2-HA, in the absence (0) or presence of increasing concentrations of PI(4,5)P2 diC8. The graph represents the band intensity of the autoradiograph signals for the WT GST-Myo5-Cext relative to the sample incubated in the absence of PI(4,5)P2 (0).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

9. Figure 8
Model for the Coordinated and Cooperative Inhibition of Myo5/Vrp1-Induced Arp2/3-Dependent Actin Polymerization by CK2 and the Synaptojanins Sjl1 and Sjl2
The cytosolic myosin-I Myo5 exists in an autoinhibited conformation stabilized by calmodulin (Cmd1; Grötsch et al., 2010). Binding of PI(4,5)P2 to the myosin lipid binding domain triggers Cmd1 dissociation and promotes Myo5 binding to the yeast WIP Vrp1. The Myo5/Vrp1 complex can then promote Arp2/3-dependent actin polymerization at endocytic sites. Hydrolysis of PI(4,5)P2 by the endocytic synaptojanins Sjl1 and Sjl2 then terminates the positive signal that releases Myo5 autoinhibition and liberates a membrane-associated monomeric pool of Cka2, the CK2α′ catalytic subunit of the tetrameric CK2. The monomeric Cka2 can then phosphorylate the Myo5 S1205, and possibly other endocytic targets, to cooperatively terminate Arp2/3-dependent actin polymerization and to promote Myo5 disassembly from the plasma membrane. This event is required for efficient constriction of the invagination neck. Binding of the regulatory CK2β subunits to Cka2 prevents phosphorylation of the cytosolic Myo5 by Cka2. Binding of PI(4,5)P2 to the catalytic subunits of CK2 favors disassembly of the holoenzyme but keeps the membrane-associated Cka2 inactive until arrival of the synaptojanins at the endocytic sites.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions