Journal: International Journal of Molecular Sciences

Article Title: Drebrin Regulates Acetylcholine Receptor Clustering and Organization of Microtubules at the Postsynaptic Machinery

doi: 10.3390/ijms22179387

Figure Lengend Snippet: Drebrin and microtubule-associated EB3 interact in myotubes and drebrin knockdown impacts microtubule capture at AChR clusters. ( A ) Co-immunoprecipitation of endogenous drebrin with overexpressed EB3-GFP in homogenates from C2C12 myotubes cultured on laminin-111. Flow-through—lysate incubated with the beads, IP—eluate from the beads coated with anti-GFP antibody, Control IP—eluate from the uncoated beads. ( B ) Control and drebrin-depleted C2C12 myotubes labeled for AChRs (green) and microtubules plus-end protein EB3 (red). Representative images from control and drebrin knockdown with siRNA1 are shown. Control myotubes were transfected with non-targeting siRNA. Scale bars—10 μm. ( C ) Quantification of EB3 foci localized underneath AChR clusters after drebrin knockdown. ( D ) EB3 foci under AChR clusters in control and BTP2-treated C2C12 myotubes labeled for microtubules plus-end protein EB3 (red) and AChRs (green). Scale bars—10 μm. ( E ) Number of EB3 foci localized underneath AChR clusters upon BTP2 treatment. Data are presented as a percentage of control (myotubes transfected with non-targeting siRNA or myotubes incubated with DMSO (vehicle)). Error bars represent SEM. Student’s T -test, p ≤ 0.001 (***); ns—not significant.

Article Snippet: The primary antibodies were as follows: anti-drebrin (10260-1-AP, ProteinTech, and 25770-1-AP, ProteinTech), anti-phospho-drebrin (MABN833, Merck), anti-EB3 (612156, BD Biosciences, USA), anti-myosin heavy chain (AB_2147781, DSHB, USA), anti-α-actinin (sc-17829, Santa Cruz Biotechnology), anti-Ryr1 (ab2868, Abcam, USA), anti-neurofilament (AB_2314897, DSHB), and anti-synaptophysin 1 (101 004, Synaptic Systems, Germany).

Techniques: Immunoprecipitation, Cell Culture, Incubation, Labeling, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Drebrin Regulates Acetylcholine Receptor Clustering and Organization of Microtubules at the Postsynaptic Machinery

doi: 10.3390/ijms22179387

Figure Lengend Snippet: BTP2 impairs the organization of AChR cluster-associated mictrotubules. ( A ) Images used for processing in Ilastik and CellProfiler—segmentation and object identification. The last image on the right is an example of images used for object size and shape measurements (see Materials and Methods, ). ( B ) Automated measurement of size and shape parameters of AChR cluster-associated EB3 foci after BTP2 treatment (CellProfiler). ( C ) Quantification of the total number of coalescent vs. single EB3 foci upon BTP2 treatment based on the surface manual measurements with ZEN software. Average surface of a single foci was used as a threshold and EB3 puncta with bigger surface were classified as coalescent. The distribution of coalescent EB3 foci is significantly different from the uniform distribution in both groups. Data are presented as a percentage of control (myotubes incubated with DMSO (vehicle)) except in ( C ), which represents a percentage of total EB3 foci analyzed in each group. Error bars represent SEM. ( B ) Student’s T -test, p ≤ 0.01 (**); p ≤ 0.001 (***); ( C ) chi-square test, p < 0.001, chi square = 681.727, 4 degrees of freedom.

Article Snippet: The primary antibodies were as follows: anti-drebrin (10260-1-AP, ProteinTech, and 25770-1-AP, ProteinTech), anti-phospho-drebrin (MABN833, Merck), anti-EB3 (612156, BD Biosciences, USA), anti-myosin heavy chain (AB_2147781, DSHB, USA), anti-α-actinin (sc-17829, Santa Cruz Biotechnology), anti-Ryr1 (ab2868, Abcam, USA), anti-neurofilament (AB_2314897, DSHB), and anti-synaptophysin 1 (101 004, Synaptic Systems, Germany).

Techniques: Software, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Drebrin Regulates Acetylcholine Receptor Clustering and Organization of Microtubules at the Postsynaptic Machinery

doi: 10.3390/ijms22179387

Figure Lengend Snippet: AChR-clustering regulator rapsyn interacts with drebrin. ( A ) Coimmunoprecipitation of overexpressed wild-type drebrin (DbnE-WT-GFP) with rapsyn (Rapsyn-mCherry) in HEK293 cells. ( B ) Coimmunoprecipitation of overexpressed phospho-dead drebrin mutant at Ser142 (DbnE-142A-GFP) with rapsyn (Rapsyn-mCherry) in HEK293 cells. ( C ) Coimmunoprecipitation of overexpressed EB3-GFP with rapsyn (Rapsyn-mCherry) in HEK293 cells. Elute—eluates from the beads incubated with equal volumes of lysates, Flow-through (FT)—lysates incubated with the beads.

Article Snippet: The primary antibodies were as follows: anti-drebrin (10260-1-AP, ProteinTech, and 25770-1-AP, ProteinTech), anti-phospho-drebrin (MABN833, Merck), anti-EB3 (612156, BD Biosciences, USA), anti-myosin heavy chain (AB_2147781, DSHB, USA), anti-α-actinin (sc-17829, Santa Cruz Biotechnology), anti-Ryr1 (ab2868, Abcam, USA), anti-neurofilament (AB_2314897, DSHB), and anti-synaptophysin 1 (101 004, Synaptic Systems, Germany).

Techniques: Mutagenesis, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Drebrin Regulates Acetylcholine Receptor Clustering and Organization of Microtubules at the Postsynaptic Machinery

doi: 10.3390/ijms22179387

Figure Lengend Snippet: Proposed model of drebrin role in the regulation of postsynaptic machinery clustering. Rapsyn anchors AChRs to actin and microtubules (− Drebrin). Drebrin bundles actin filaments and acts as a cross-linker between actin cytoskeleton and microtubule network under AChR clusters. Drebrin interacts with rapsyn, a cluster-stabilizing protein, and EB3, a microtubule plus-end protein, and recruits microtubules under AChR clusters (+ Drebrin). Illustration created with BioRender.com ( www.biorender.com , last access 26 August 2021).

Article Snippet: The primary antibodies were as follows: anti-drebrin (10260-1-AP, ProteinTech, and 25770-1-AP, ProteinTech), anti-phospho-drebrin (MABN833, Merck), anti-EB3 (612156, BD Biosciences, USA), anti-myosin heavy chain (AB_2147781, DSHB, USA), anti-α-actinin (sc-17829, Santa Cruz Biotechnology), anti-Ryr1 (ab2868, Abcam, USA), anti-neurofilament (AB_2314897, DSHB), and anti-synaptophysin 1 (101 004, Synaptic Systems, Germany).

Techniques: