Journal: bioRxiv

Article Title: Cluster nanoarchitecture and structural diversity of PIEZO1 in intact cells

doi: 10.1101/2024.11.26.625366

Figure Lengend Snippet: a , cryo-EM structure of PIEZO and localization of mGreenLantern(mGL)-tag (left) and cartoon illustrating the principle of MINFLUX/DNA-PAINT (right). DNA-PAINT utilizes complementary pairs of short oligonucleotides, an imager strand conjugated to the fluorophore and a docking strand conjugated to an sd-Nb that binds to the target. To determine the fluorophore position, the MINFLUX microscope performs an iterative 3D scanning procedure, which is repeated until the fluorophore is bleached or the imager strand dissociates from the target, such that ‘traces’ with multiple localization estimates of the same fluorophore are generated. b , shows a confocal scan of an N2a cell expressing PIEZO1-mGL and c , shows the corresponding MINFLUX localization data (left) and the distribution of the standard deviations of the MINFLUX traces along the x, y, and z-axis (right) d and e , show 3D-views, top views and side views of the raw MINFLUX localization data of the pit-shaped (d) and the spherical (e) clusters marked in c . For 360° rotation movies of the clusters shown in d and e, see Supplementary Video 1 and 2. f , Overlay of the 2D projections of the traces means of the top fifth and bottom four fifth of all pit-shaped clusters (blue circles, N=158 cluster from 19 cells) and spherical clusters (grey circles, N=93 clusters from 19 cells). Note, in pit-shaped clusters no channels are present at the center of the top fifth. g , bar graph showing the proportions of pit-shaped and spherical clusters.

Article Snippet: PIEZO1-mGreenLantern fusion protein (PIEZO1-mGL) was generated by amplifying the coding sequence of the green fluorescent protein mGreenLantern from a LifeAct-mGreenLantern plasmid (git from G. Petsko, Addgene #164459), and inserted with a SG linker after the C-ter of PIEZO1 plasmid, where the IRES-GFP has sequence has been excised beforehand by PCR.

Techniques: Cryo-EM Sample Prep, Microscopy, Generated, Expressing

Journal: bioRxiv

Article Title: Cluster nanoarchitecture and structural diversity of PIEZO1 in intact cells

doi: 10.1101/2024.11.26.625366

Figure Lengend Snippet: a , raw MINFLUX localization data color coded by Z-position of clusters imaged in control cells (top) and cells challenged with hypoosmotic solution (120 mOsm) before fixation (bottom). b , 3D-views, of the raw MINFLUX localization data of representative clusters from control (top) and osmotically stimulated (bottom) cells together with surface fits. For 360° rotation movies of the clusters see Supplementary Video 4-5. c , comparison of the cluster depths of control and hypo-osmotically stimulated cells (CTL: 144 ± 60 nm, N=158 vs. OSMO: 110.7 ± 54 nm, N=79, P=0.000052, two-tailed Student’s t-test). d , comparison of the proportions of pit-shaped (blue) and spherical clusters (grey) in control (CTL) and hypo-osmotically stimulated (OSMO) cells. The numbers provided in the bars represent the absolute number of clusters from the two categories. e , comparison of the mean ± sem number of PIEZO1 channel found in pit-shaped clusters. Circles represent the number of channels in individual clusters (CTL: 23.1±11.3, N=158 vs. OSMO: 18.7±8.0, N=79, P=0.0134, two-tailed Mann-Whitney test). f , histograms showing the distribution of the radii of all pit-shaped clusters measured at the pit opening. Medians are indicated by the red dashed lines. g , the left panel shows the side view of an example surface fit in which the contours of convex regions are depicted in blue and the contours of concave regions are shown in yellow. Gaussian curvatures of the fitted surfaces (see b) at the coordinates where channels were detected, were calculated and are shown as horizontal box plots. Boxes range from the lower quartile to the upper quartile, medians are shown as red lines and negative and positive curvatures are color coded in blue and yellow, respectively. Mean curvatures were compared using non-parametric Mann Whitney test (***, P = 0.0004, N=3550 for CTL and N=1358 for OSMO).

Article Snippet: PIEZO1-mGreenLantern fusion protein (PIEZO1-mGL) was generated by amplifying the coding sequence of the green fluorescent protein mGreenLantern from a LifeAct-mGreenLantern plasmid (git from G. Petsko, Addgene #164459), and inserted with a SG linker after the C-ter of PIEZO1 plasmid, where the IRES-GFP has sequence has been excised beforehand by PCR.

Techniques: Control, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cluster nanoarchitecture and structural diversity of PIEZO1 in intact cells

doi: 10.1101/2024.11.26.625366

Figure Lengend Snippet: a , side and top views of PIEZO, in the curved (grey) and flattened (red) conformation. Distances between ALFA-tags from one trimer are designated as ‘interblade distance’ and the angles between the lines connecting the ALFA-tags (α,β,γ) as ‘interblade angles’. b , confocal image of an N2a cell expressing PIEZO1-ALFA-mGL (left) and top and side view of the corresponding MINFLUX localization data from the marked regions in the soma and neurites (Supplementary Video 6 for 360° rotation movies of the example shown here and Supplementary Video 7-10 for additional examples). c , 3D scatter plot of raw localizations of representative trimers found in a cell soma (top-left) and in a neurite (bottom-left). Localizations originating from fluorophores bound to different protomers are colored in black, dark grey and light grey. The 3D raw localizations were projected vertically onto the plane defined by the trace means (2D in-plane projection) and then fitted with a bivariate Gaussian distribution. The heatmaps in the right column show the probability densities of the Gaussian distributions. d , superparticle of all trimers found in somata (top) and in neurites (bottom), generated by aligning the trace means of each identified trimer to a reference trimer (equilateral triangle with a side length of 25nm) using the iterative closest point algorithm of Matlab. e , comparison of the mean ± sem interblade distances of trimers residing in somata (black bar, N=67 trimers from 10 cells) and neurites (grey bar, N=30 trimers from 10 cells) using two-tailed Students t-test (P=0.00019). Values from individual trimers are shown as grey circles. f , Interblade distance are plotted against neurite diameter, showing that there is no correlation between the two parameters (Pearson coefficient r = 0.153). g , cartoon showing the lack of an effect of membrane curvature on PIEZO1 conformation and highlighting differences in cytoskeletal architecture of somata and neurites.

Article Snippet: PIEZO1-mGreenLantern fusion protein (PIEZO1-mGL) was generated by amplifying the coding sequence of the green fluorescent protein mGreenLantern from a LifeAct-mGreenLantern plasmid (git from G. Petsko, Addgene #164459), and inserted with a SG linker after the C-ter of PIEZO1 plasmid, where the IRES-GFP has sequence has been excised beforehand by PCR.

Techniques: Expressing, Generated, Comparison, Two Tailed Test, Membrane

Journal: bioRxiv

Article Title: Cluster nanoarchitecture and structural diversity of PIEZO1 in intact cells

doi: 10.1101/2024.11.26.625366

Figure Lengend Snippet: a , cartoon showing the possible interplay of forces. PIEZO1 exerts a bending force onto the membrane (F PIEZO1 , F p ) while the membrane (F membrane , F m ) and the cytoskeleton (F cytoskeleton , F c ) exert opposing forces on PIEZO1, which work towards opening the channel. We hypothesize that the equilibrium of these forces eventually determines PIEZO1 conformation. The bottom panel illustrates the effect of cytochalasin-D on the actin cortex and the resulting change in the force equilibrium. b , example traces of PIEZO1-mediated currents evoked by negative pressure in cell-attached patch-clamp recordings. c , left panel shows normalized (I/Imax) pressure-response curves of PIEZO1 currents recorded from control (black circles) and cyto-D treated (red circles) cells (left panel). Comparison of the mean ± sem P50 values (P50 = pressure required for half-maximal activation, right panel) obtained by Boltzmann fits of the data shown in the left panel, using Student’s t-test (CTL: p50 = -40.6 ± 2.5 mmHg, N=20 vs. cyto-D: p50 = -29.1 ± 2.6 mmHg, N =16; P = 0.0038). White circles show P50 values from individual recordings. d , side views of MINFLUX raw localization data from a control (top) and a cyto-D-treated cell. Note, cyto-D treatment does not change the curvature of the cell-substrate interface. e , 2-D in-plane projections of the raw localization data (left) and heatmaps of the localization probability densities (right) of representative trimers from control (CTL, top) and cyto-D (bottom) treated cells. f , superparticle of all trimers found in control (CTL, top) and cyto-D (bottom) treated cells (see description in ). g , comparison of the mean ± sem interblade distances of all trimers from control (23.90 ± 5.14 nm, N=67, grey) and cyto-D (20.58 ± 5.30, N=53, red) treated cells using Students t-test (P=0.0007). Values from individual trimers are shown as grey circles.

Article Snippet: PIEZO1-mGreenLantern fusion protein (PIEZO1-mGL) was generated by amplifying the coding sequence of the green fluorescent protein mGreenLantern from a LifeAct-mGreenLantern plasmid (git from G. Petsko, Addgene #164459), and inserted with a SG linker after the C-ter of PIEZO1 plasmid, where the IRES-GFP has sequence has been excised beforehand by PCR.

Techniques: Membrane, Patch Clamp, Control, Comparison, Activation Assay

Journal: The Journal of Neuroscience

Article Title: CDK5/p35-Dependent Microtubule Reorganization Contributes to Homeostatic Shortening of the Axon Initial Segment

doi: 10.1523/jneurosci.0917-22.2022

Figure Lengend Snippet: Figure 6. AIS shortening occurred in a manner dependent on CDK5 activity. A, Time course of the experiments. Plasmids were introduced into NM neurons at E2, HCF slices were prepared at E 11, and DOX was added to the culture medium at 6–10 DIV. B, tdTomato (red) was expressed in NM neurons (ipsi) in slice culture stained with panNav antibody (white). Dotted line indi- cates the midline. C–K, AIS of NM neurons with (ipsi) or without (contra) overexpression of dnCDK5 in 2 [K1] medium (C), and of CDK5 (E), p35 (G), p35(T138A) (I), or CDK5 and p35 (K) in normal (1 [K1]) medium. Plasmids used are shown in C–K. Note the absence of Nav signals at AIS in CDK5 and p35 double-positive neurons (K, left). Intensity profiles of Nav signal are the average of 10 cells (D, F, H, J). L, M, Length of AIS in 2 [K1] (L) and 1 [K1] (M) media. Numbers in parentheses indicate the number of cells. N, Ratio of mRNA level of CDK5 and p35 between 2 [K1] and 1 [K1] media. Numbers in parentheses represent the number of experiments in N. O, AIS length of NM neurons from LCF with (ipsi) or without (contra) overex- pression of p35 in normal (1 [K1]) medium. Plasmid in G was used; *p , 0.05, **p , 0.01 compared with mock by Student’s t test (L, N) and one-way ANOVA and post hoc test (M). Ipsi, Ipsilateral; contra, contralateral.

Article Snippet: All plasmids were constructed by inserting the following sequences into the plasmid backbone of pCAG-floxedSTOP-tdTomato-WPRE (Egawa and Yawo, 2019) using In-Fusion cloning (Takara Bio): hSyn1 promoter (pLenti Syn hChR2-EYFP-Nav1.2II-III; a gift from Matthew S. Grubb; Grubb and Burrone, 2010), TetOn3G and TRE3GS promoter (pTetOne Vector, catalog #634301, Clontech), mGreenLantern (LifeAct-mGreenLantern, plasmid catalog #164459, Addgene; a gift from Gregory Petsko; Campbell et al., 2020), CDK5 and dominant-negative CDK5 (Cdk5-HA and Cdk5DN-HA, plasmid #1872 and #1873, Addgene; a gift from Sander van den Heuvel; van den Heuvel and Harlow, 1993), and p35 (pCMV-P35, plasmid #1347, Addgene; a gift from Li-Huei Tsai).

Techniques: Activity Assay, Staining, Over Expression, Plasmid Preparation

Journal: The Journal of Neuroscience

Article Title: CDK5/p35-Dependent Microtubule Reorganization Contributes to Homeostatic Shortening of the Axon Initial Segment

doi: 10.1523/jneurosci.0917-22.2022

Figure Lengend Snippet: Figure 7. CDK5 mediated AIS shortening via reorganization of microtubules. A, Time course of the experiments. HCF slices were incubated with stabilizers of microtubules (MT) or actin dur- ing treatment with 2 [K1] medium, FSK or PMA, or okadaic acid for 7–10 DIV. B, Pharmacological manipulation of microtubule dynamics. C, Effects of MT and actin filament stabilizers on AIS of NM neurons in 2 [K1] medium. D, E, MT stabilizers occluded AIS shortening by 2 [K1] medium (D, left), by FSK, PMA (D, right), or by okadaic acid (E). AIS lengths for 2 [K1] (green), FSK or PMA alone (light gray), and normal (1 [K1]) medium (light gray) were from Figures 2I, 5D, and 1F (10 DIV), respectively; **p , 0.01 compared with 2 [K1] (D, left), 1 [K1] (E) by one-way ANOVA and post hoc test, FSK or PMA alone (D, right) by Kruskal–Wallis test. F–I, Taxol occluded AIS shortening by overexpression of p35 (G) or p35 together with CDK5 (H). Time course of experiments (F) and AIS length (I). p35 and p35 together with CDK5 are from Figure 6M; **p , 0.01 by Student’s t test. Numbers in parentheses indicate the number of cells.

Article Snippet: All plasmids were constructed by inserting the following sequences into the plasmid backbone of pCAG-floxedSTOP-tdTomato-WPRE (Egawa and Yawo, 2019) using In-Fusion cloning (Takara Bio): hSyn1 promoter (pLenti Syn hChR2-EYFP-Nav1.2II-III; a gift from Matthew S. Grubb; Grubb and Burrone, 2010), TetOn3G and TRE3GS promoter (pTetOne Vector, catalog #634301, Clontech), mGreenLantern (LifeAct-mGreenLantern, plasmid catalog #164459, Addgene; a gift from Gregory Petsko; Campbell et al., 2020), CDK5 and dominant-negative CDK5 (Cdk5-HA and Cdk5DN-HA, plasmid #1872 and #1873, Addgene; a gift from Sander van den Heuvel; van den Heuvel and Harlow, 1993), and p35 (pCMV-P35, plasmid #1347, Addgene; a gift from Li-Huei Tsai).

Techniques: Incubation, Over Expression

Journal: The Journal of Neuroscience

Article Title: CDK5/p35-Dependent Microtubule Reorganization Contributes to Homeostatic Shortening of the Axon Initial Segment

doi: 10.1523/jneurosci.0917-22.2022

Figure Lengend Snippet: Figure 5. AIS shortening occurred via activation of MEK and CDK5 pathways. A, Time course of the experiments. HCF slices were incubated with kinase inhibitors in a 2 [K1] medium or with activators in a normal (1 [K1]) medium for 7–10 DIV. B, MEK signaling pathway. C, AIS of NM neurons. D–F, Length of AIS. Effects of kinase inhibitors (D), concentration dependence of U0126 (E), and phosphatase inhibitors (F), respectively in the 2 [K1] medium. Control (2 [K1], green) is from Figure 2I. G, Activators of PKA and PKC shortened AIS in the 1 [K1] medium. Control (1 [K1], light gray) is from Figure 1F. H, MEK or CDK5 inhibitors occluded AIS shortening by forskolin (FSK) or PMA. AIS lengths for FSK and PMA alone (light gray) are from Figure 5G. Numbers in parentheses indicate the number of cells; *p , 0.05, **p , 0.01 compared with 2 [K1] (D–F) by one-way ANOVA and post hoc test, 1 [K1] (G), and FSK or PMA alone (H) by Kruskal–Wallis test.

Article Snippet: All plasmids were constructed by inserting the following sequences into the plasmid backbone of pCAG-floxedSTOP-tdTomato-WPRE (Egawa and Yawo, 2019) using In-Fusion cloning (Takara Bio): hSyn1 promoter (pLenti Syn hChR2-EYFP-Nav1.2II-III; a gift from Matthew S. Grubb; Grubb and Burrone, 2010), TetOn3G and TRE3GS promoter (pTetOne Vector, catalog #634301, Clontech), mGreenLantern (LifeAct-mGreenLantern, plasmid catalog #164459, Addgene; a gift from Gregory Petsko; Campbell et al., 2020), CDK5 and dominant-negative CDK5 (Cdk5-HA and Cdk5DN-HA, plasmid #1872 and #1873, Addgene; a gift from Sander van den Heuvel; van den Heuvel and Harlow, 1993), and p35 (pCMV-P35, plasmid #1347, Addgene; a gift from Li-Huei Tsai).

Techniques: Activation Assay, Incubation, Concentration Assay, Control

Journal: The Journal of Neuroscience

Article Title: CDK5/p35-Dependent Microtubule Reorganization Contributes to Homeostatic Shortening of the Axon Initial Segment

doi: 10.1523/jneurosci.0917-22.2022

Figure Lengend Snippet: Figure 6. AIS shortening occurred in a manner dependent on CDK5 activity. A, Time course of the experiments. Plasmids were introduced into NM neurons at E2, HCF slices were prepared at E 11, and DOX was added to the culture medium at 6–10 DIV. B, tdTomato (red) was expressed in NM neurons (ipsi) in slice culture stained with panNav antibody (white). Dotted line indi- cates the midline. C–K, AIS of NM neurons with (ipsi) or without (contra) overexpression of dnCDK5 in 2 [K1] medium (C), and of CDK5 (E), p35 (G), p35(T138A) (I), or CDK5 and p35 (K) in normal (1 [K1]) medium. Plasmids used are shown in C–K. Note the absence of Nav signals at AIS in CDK5 and p35 double-positive neurons (K, left). Intensity profiles of Nav signal are the average of 10 cells (D, F, H, J). L, M, Length of AIS in 2 [K1] (L) and 1 [K1] (M) media. Numbers in parentheses indicate the number of cells. N, Ratio of mRNA level of CDK5 and p35 between 2 [K1] and 1 [K1] media. Numbers in parentheses represent the number of experiments in N. O, AIS length of NM neurons from LCF with (ipsi) or without (contra) overex- pression of p35 in normal (1 [K1]) medium. Plasmid in G was used; *p , 0.05, **p , 0.01 compared with mock by Student’s t test (L, N) and one-way ANOVA and post hoc test (M). Ipsi, Ipsilateral; contra, contralateral.

Article Snippet: All plasmids were constructed by inserting the following sequences into the plasmid backbone of pCAG-floxedSTOP-tdTomato-WPRE (Egawa and Yawo, 2019) using In-Fusion cloning (Takara Bio): hSyn1 promoter (pLenti Syn hChR2-EYFP-Nav1.2II-III; a gift from Matthew S. Grubb; Grubb and Burrone, 2010), TetOn3G and TRE3GS promoter (pTetOne Vector, catalog #634301, Clontech), mGreenLantern (LifeAct-mGreenLantern, plasmid catalog #164459, Addgene; a gift from Gregory Petsko; Campbell et al., 2020), CDK5 and dominant-negative CDK5 (Cdk5-HA and Cdk5DN-HA, plasmid #1872 and #1873, Addgene; a gift from Sander van den Heuvel; van den Heuvel and Harlow, 1993), and p35 (pCMV-P35, plasmid #1347, Addgene; a gift from Li-Huei Tsai).

Techniques: Activity Assay, Staining, Over Expression, Plasmid Preparation

Journal: The Journal of Neuroscience

Article Title: CDK5/p35-Dependent Microtubule Reorganization Contributes to Homeostatic Shortening of the Axon Initial Segment

doi: 10.1523/jneurosci.0917-22.2022

Figure Lengend Snippet: Figure 7. CDK5 mediated AIS shortening via reorganization of microtubules. A, Time course of the experiments. HCF slices were incubated with stabilizers of microtubules (MT) or actin dur- ing treatment with 2 [K1] medium, FSK or PMA, or okadaic acid for 7–10 DIV. B, Pharmacological manipulation of microtubule dynamics. C, Effects of MT and actin filament stabilizers on AIS of NM neurons in 2 [K1] medium. D, E, MT stabilizers occluded AIS shortening by 2 [K1] medium (D, left), by FSK, PMA (D, right), or by okadaic acid (E). AIS lengths for 2 [K1] (green), FSK or PMA alone (light gray), and normal (1 [K1]) medium (light gray) were from Figures 2I, 5D, and 1F (10 DIV), respectively; **p , 0.01 compared with 2 [K1] (D, left), 1 [K1] (E) by one-way ANOVA and post hoc test, FSK or PMA alone (D, right) by Kruskal–Wallis test. F–I, Taxol occluded AIS shortening by overexpression of p35 (G) or p35 together with CDK5 (H). Time course of experiments (F) and AIS length (I). p35 and p35 together with CDK5 are from Figure 6M; **p , 0.01 by Student’s t test. Numbers in parentheses indicate the number of cells.

Article Snippet: All plasmids were constructed by inserting the following sequences into the plasmid backbone of pCAG-floxedSTOP-tdTomato-WPRE (Egawa and Yawo, 2019) using In-Fusion cloning (Takara Bio): hSyn1 promoter (pLenti Syn hChR2-EYFP-Nav1.2II-III; a gift from Matthew S. Grubb; Grubb and Burrone, 2010), TetOn3G and TRE3GS promoter (pTetOne Vector, catalog #634301, Clontech), mGreenLantern (LifeAct-mGreenLantern, plasmid catalog #164459, Addgene; a gift from Gregory Petsko; Campbell et al., 2020), CDK5 and dominant-negative CDK5 (Cdk5-HA and Cdk5DN-HA, plasmid #1872 and #1873, Addgene; a gift from Sander van den Heuvel; van den Heuvel and Harlow, 1993), and p35 (pCMV-P35, plasmid #1347, Addgene; a gift from Li-Huei Tsai).

Techniques: Incubation, Over Expression

Journal: The Journal of Neuroscience

Article Title: CDK5/p35-Dependent Microtubule Reorganization Contributes to Homeostatic Shortening of the Axon Initial Segment

doi: 10.1523/jneurosci.0917-22.2022

Figure Lengend Snippet: Figure 5. AIS shortening occurred via activation of MEK and CDK5 pathways. A, Time course of the experiments. HCF slices were incubated with kinase inhibitors in a 2 [K1] medium or with activators in a normal (1 [K1]) medium for 7–10 DIV. B, MEK signaling pathway. C, AIS of NM neurons. D–F, Length of AIS. Effects of kinase inhibitors (D), concentration dependence of U0126 (E), and phosphatase inhibitors (F), respectively in the 2 [K1] medium. Control (2 [K1], green) is from Figure 2I. G, Activators of PKA and PKC shortened AIS in the 1 [K1] medium. Control (1 [K1], light gray) is from Figure 1F. H, MEK or CDK5 inhibitors occluded AIS shortening by forskolin (FSK) or PMA. AIS lengths for FSK and PMA alone (light gray) are from Figure 5G. Numbers in parentheses indicate the number of cells; *p , 0.05, **p , 0.01 compared with 2 [K1] (D–F) by one-way ANOVA and post hoc test, 1 [K1] (G), and FSK or PMA alone (H) by Kruskal–Wallis test.

Article Snippet: All plasmids were constructed by inserting the following sequences into the plasmid backbone of pCAG-floxedSTOP-tdTomato-WPRE (Egawa and Yawo, 2019) using In-Fusion cloning (Takara Bio): hSyn1 promoter (pLenti Syn hChR2-EYFP-Nav1.2II-III; a gift from Matthew S. Grubb; Grubb and Burrone, 2010), TetOn3G and TRE3GS promoter (pTetOne Vector, catalog #634301, Clontech), mGreenLantern (LifeAct-mGreenLantern, plasmid catalog #164459, Addgene; a gift from Gregory Petsko; Campbell et al., 2020), CDK5 and dominant-negative CDK5 (Cdk5-HA and Cdk5DN-HA, plasmid #1872 and #1873, Addgene; a gift from Sander van den Heuvel; van den Heuvel and Harlow, 1993), and p35 (pCMV-P35, plasmid #1347, Addgene; a gift from Li-Huei Tsai).

Techniques: Activation Assay, Incubation, Concentration Assay, Control

Journal: The Journal of Neuroscience

Article Title: CDK5/p35-Dependent Microtubule Reorganization Contributes to Homeostatic Shortening of the Axon Initial Segment

doi: 10.1523/jneurosci.0917-22.2022

Figure Lengend Snippet: Figure 6. AIS shortening occurred in a manner dependent on CDK5 activity. A, Time course of the experiments. Plasmids were introduced into NM neurons at E2, HCF slices were prepared at E 11, and DOX was added to the culture medium at 6–10 DIV. B, tdTomato (red) was expressed in NM neurons (ipsi) in slice culture stained with panNav antibody (white). Dotted line indi- cates the midline. C–K, AIS of NM neurons with (ipsi) or without (contra) overexpression of dnCDK5 in 2 [K1] medium (C), and of CDK5 (E), p35 (G), p35(T138A) (I), or CDK5 and p35 (K) in normal (1 [K1]) medium. Plasmids used are shown in C–K. Note the absence of Nav signals at AIS in CDK5 and p35 double-positive neurons (K, left). Intensity profiles of Nav signal are the average of 10 cells (D, F, H, J). L, M, Length of AIS in 2 [K1] (L) and 1 [K1] (M) media. Numbers in parentheses indicate the number of cells. N, Ratio of mRNA level of CDK5 and p35 between 2 [K1] and 1 [K1] media. Numbers in parentheses represent the number of experiments in N. O, AIS length of NM neurons from LCF with (ipsi) or without (contra) overex- pression of p35 in normal (1 [K1]) medium. Plasmid in G was used; *p , 0.05, **p , 0.01 compared with mock by Student’s t test (L, N) and one-way ANOVA and post hoc test (M). Ipsi, Ipsilateral; contra, contralateral.

Article Snippet: All plasmids were constructed by inserting the following sequences into the plasmid backbone of pCAG-floxedSTOP-tdTomato-WPRE (Egawa and Yawo, 2019) using In-Fusion cloning (Takara Bio): hSyn1 promoter (pLenti Syn hChR2-EYFP-Nav1.2II-III; a gift from Matthew S. Grubb; Grubb and Burrone, 2010), TetOn3G and TRE3GS promoter (pTetOne Vector, catalog #634301, Clontech), mGreenLantern (LifeAct-mGreenLantern, plasmid catalog #164459, Addgene; a gift from Gregory Petsko; Campbell et al., 2020), CDK5 and dominant-negative CDK5 (Cdk5-HA and Cdk5DN-HA, plasmid #1872 and #1873, Addgene; a gift from Sander van den Heuvel; van den Heuvel and Harlow, 1993), and p35 (pCMV-P35, plasmid #1347, Addgene; a gift from Li-Huei Tsai).

Techniques: Activity Assay, Staining, Over Expression, Plasmid Preparation

Journal: The Journal of Neuroscience

Article Title: CDK5/p35-Dependent Microtubule Reorganization Contributes to Homeostatic Shortening of the Axon Initial Segment

doi: 10.1523/jneurosci.0917-22.2022

Figure Lengend Snippet: Figure 7. CDK5 mediated AIS shortening via reorganization of microtubules. A, Time course of the experiments. HCF slices were incubated with stabilizers of microtubules (MT) or actin dur- ing treatment with 2 [K1] medium, FSK or PMA, or okadaic acid for 7–10 DIV. B, Pharmacological manipulation of microtubule dynamics. C, Effects of MT and actin filament stabilizers on AIS of NM neurons in 2 [K1] medium. D, E, MT stabilizers occluded AIS shortening by 2 [K1] medium (D, left), by FSK, PMA (D, right), or by okadaic acid (E). AIS lengths for 2 [K1] (green), FSK or PMA alone (light gray), and normal (1 [K1]) medium (light gray) were from Figures 2I, 5D, and 1F (10 DIV), respectively; **p , 0.01 compared with 2 [K1] (D, left), 1 [K1] (E) by one-way ANOVA and post hoc test, FSK or PMA alone (D, right) by Kruskal–Wallis test. F–I, Taxol occluded AIS shortening by overexpression of p35 (G) or p35 together with CDK5 (H). Time course of experiments (F) and AIS length (I). p35 and p35 together with CDK5 are from Figure 6M; **p , 0.01 by Student’s t test. Numbers in parentheses indicate the number of cells.

Article Snippet: All plasmids were constructed by inserting the following sequences into the plasmid backbone of pCAG-floxedSTOP-tdTomato-WPRE (Egawa and Yawo, 2019) using In-Fusion cloning (Takara Bio): hSyn1 promoter (pLenti Syn hChR2-EYFP-Nav1.2II-III; a gift from Matthew S. Grubb; Grubb and Burrone, 2010), TetOn3G and TRE3GS promoter (pTetOne Vector, catalog #634301, Clontech), mGreenLantern (LifeAct-mGreenLantern, plasmid catalog #164459, Addgene; a gift from Gregory Petsko; Campbell et al., 2020), CDK5 and dominant-negative CDK5 (Cdk5-HA and Cdk5DN-HA, plasmid #1872 and #1873, Addgene; a gift from Sander van den Heuvel; van den Heuvel and Harlow, 1993), and p35 (pCMV-P35, plasmid #1347, Addgene; a gift from Li-Huei Tsai).

Techniques: Incubation, Over Expression