imaging superresolution stochastic optical reconstruction microscopy storm imaging  (Nikon)

Journal: Neural regeneration research

Article Title: The interaction between KIF21A and KANK1 regulates dendritic morphology and synapse plasticity in neurons.

doi: 10.4103/1673-5374.391301

Figure Lengend Snippet: Figure 5 ｜The KIF21A-KANK1 interaction regulates the distribution of both proteins in dendritic spines. (A) A typical side-view synapse with Homer1c (red, Alexa 647) labeled with an antibody and imaged by stochastic optical reconstruction microscopy (STORM), and F-actin filaments represented by Lifeact-skylan and imaged with PALM. (B) Distribution of Homer1c and Lifeact density along the synaptic axis (n = 39 spines from 8 cells). Data from individual synapses were averaged within a single field of view, as shown in A, and aligned with the Homer1c distribution peaks. (C) Representative STORM/PALM images of Lifeact (green, skylan) and KIF21A (WT, red, HA, Alexa 647) or KIF21A (ΔH, red, HA), which respectively represent Lifeact with KIF21A ΔH or wild-type KIF21A (transfected with the Lifeact plasmid and the KIF21A ΔH or wild-type KIF21A plasmid). This set of plots shows that Lifeact distribution did not change significantly in the two groups, whereas the truncated KIF21A was distributed closer to the dendrites and exhibited a more diffuse distribution pattern than wild-type KIF21A. (D) Pooled distributions of these proteins along the synaptic axis (n = 73 spines from 11 cells for KIF21A (WT) and 53 spines from 10 cells for KIF21A (ΔH)). (E) Representative STORM images of Homer1c (red, Alexa 647) and KANK1 (green, CyTM3B) expression in neurons transfected with the control plasmid or the shKIF21A plasmid, which respectively represent the normal group (transfected with the control plasmid) and the shKIF21A group (transfected with the shKIF21A plasmid). This set of figures shows that the distribution of Lifeact did not change significantly in the two groups, but KIF21A knockdown resulted in more diffuse KANK1 distributing further away from the dendritic shaft compared with expression of the control shRNA. The rectangles in A, C, and E indicate the portion of the data that was analyzed. Scale bars: 200 nm. (F) Pooled distributions of Homer1c and KANK1 along the synaptic axis (n = 37 spines from 8 cells for control and 36 spines from 7 cells for shKIF21A). ctrl: Control; Homer1c: homer scaffold protein 1c; HA: Human influenza hemagglutinin; KANK1: KN motif and ankyrin repeat domain-containing protein 1; KIF21A: Kinesin Family member 21A; KIF21A (ΔH): KIF21A missing H sequence; WT: wild type.

Article Snippet: imaging Superresolution stochastic optical reconstruction microscopy (STORM) imaging was conducted using a Nikon ECLIPSE Ti2 inverted microscope equipped with a perfect focusing system and a 100×/1.49 TIRF oil-immersion objective controlled using NIS Elements AR 4.30.02 software (Tokyo, Japan).

Techniques: Labeling, Microscopy, Transfection, Plasmid Preparation, Expressing, Control, Knockdown, shRNA, Sequencing

Journal: Neural Regeneration Research

Article Title: The interaction between KIF21A and KANK1 regulates dendritic morphology and synapse plasticity in neurons

doi: 10.4103/1673-5374.391301

Figure Lengend Snippet: The KIF21A-KANK1 interaction regulates the distribution of both proteins in dendritic spines. (A) A typical side-view synapse with Homer1c (red, Alexa 647) labeled with an antibody and imaged by stochastic optical reconstruction microscopy (STORM), and F-actin filaments represented by Lifeact-skylan and imaged with PALM. (B) Distribution of Homer1c and Lifeact density along the synaptic axis ( n = 39 spines from 8 cells). Data from individual synapses were averaged within a single field of view, as shown in A, and aligned with the Homer1c distribution peaks. (C) Representative STORM/PALM images of Lifeact (green, skylan) and KIF21A (WT, red, HA, Alexa 647) or KIF21A (ΔH, red, HA), which respectively represent Lifeact with KIF21A ΔH or wild-type KIF21A (transfected with the Lifeact plasmid and the KIF21A ΔH or wild-type KIF21A plasmid). This set of plots shows that Lifeact distribution did not change significantly in the two groups, whereas the truncated KIF21A was distributed closer to the dendrites and exhibited a more diffuse distribution pattern than wild-type KIF21A. (D) Pooled distributions of these proteins along the synaptic axis ( n = 73 spines from 11 cells for KIF21A (WT) and 53 spines from 10 cells for KIF21A (ΔH)). (E) Representative STORM images of Homer1c (red, Alexa 647) and KANK1 (green, Cy TM 3B) expression in neurons transfected with the control plasmid or the shKIF21A plasmid, which respectively represent the normal group (transfected with the control plasmid) and the shKIF21A group (transfected with the shKIF21A plasmid). This set of figures shows that the distribution of Lifeact did not change significantly in the two groups, but KIF21A knockdown resulted in more diffuse KANK1 distributing further away from the dendritic shaft compared with expression of the control shRNA. The rectangles in A, C, and E indicate the portion of the data that was analyzed. Scale bars: 200 nm. (F) Pooled distributions of Homer1c and KANK1 along the synaptic axis ( n = 37 spines from 8 cells for control and 36 spines from 7 cells for shKIF21A). ctrl: Control; Homer1c: homer scaffold protein 1c; HA: Human influenza hemagglutinin; KANK1: KN motif and ankyrin repeat domain-containing protein 1; KIF21A: Kinesin Family member 21A; KIF21A (ΔH): KIF21A missing H sequence; WT: wild type.

Article Snippet: Superresolution stochastic optical reconstruction microscopy (STORM) imaging was conducted using a Nikon ECLIPSE Ti2 inverted microscope equipped with a perfect focusing system and a 100×/1.49 TIRF oil-immersion objective controlled using NIS Elements AR 4.30.02 software (Tokyo, Japan).

Techniques: Labeling, Microscopy, Transfection, Plasmid Preparation, Expressing, Control, Knockdown, shRNA, Sequencing