Specific depletion of the motor protein KIF5B leads to deficits in dendritic transport, synaptic plasticity and memory
- The University of Hong Kong, China
- Chinese University of Hong Kong, China
- Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, China
Figures
Figure 1 with 1 supplement
Different roles of KIF5s on dendritic spine morphogenesis and synaptic function.
(A) Western blot showing the developmental expression of different Kinesin I motors in the mouse hippocampus. The expression of all three KIF5s significantly decreased at later postnatal stages as …
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Figure 1—source data 1
Plotted values for Figure 1.
- https://cdn.elifesciences.org/articles/53456/elife-53456-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Validation of the knockdown efficiency and specificity of different shRNAs targeting KIF5.
(A) For western blot analysis, cortical neurons were transfected with the different shRNAs by nucleofection. At DIV 5, lysate was collected for Western blotting with antibody that specifically …
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Figure 1—figure supplement 1—source data 1
Plotted values for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/53456/elife-53456-fig1-figsupp1-data1-v2.xlsx
Figure 2 with 1 supplement
Non-redundant roles of KIF5A and KIF5B on dendritic spine morphogenesis.
(A) Representative images of dissociated rat hippocampal neurons co-transfected with GFP and KIF5B-shRNA with/without KIF5B or KIF5A construct. Co-expression of KIF5B rescued the loss of mushroom …
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Figure 2—source data 1
Plotted values for Figure 2.
- https://cdn.elifesciences.org/articles/53456/elife-53456-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Subcellular localization of KIF5A and KIF5B in hippocampal neurons.
(A) Dissociated hippocampal neurons were transfected with tdTomato and equal amounts of either KIF5A-GFP or KIF5B-GFP. Greyscale images for individual channels, 2-channel merged images (blue for …
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Figure 2—figure supplement 1—source data 1
Plotted values for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/53456/elife-53456-fig2-figsupp1-data1-v2.xlsx
Figure 3 with 1 supplement
KIF5A and KIF5B differentially regulate the dendritic transport of FMRP and mRNA cargoes.
(A) Schematic diagram of the motor, stalk and tail domains of KIF5A, KIF5B and KIF5C, with the corresponding amino-acid positions indicated. The diverse carboxyl-termini within the tail domains were …
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Figure 3—source data 1
Plotted values for Figure 3.
- https://cdn.elifesciences.org/articles/53456/elife-53456-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Quantification of GFP-FMRP granule motility and percentage of movements after knockdown of KIF5A or KIF5B.
(A) Demonstration of GFP-FMRP granule quantification using kymograph tracing. Different types of movements were traced manually and displayed in different colors. (Stat: stationary; Osc: oscillatory;…
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Figure 3—figure supplement 1—source data 1
Plotted values for Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/53456/elife-53456-fig3-figsupp1-data1-v2.xlsx
Figure 4
Carboxyl-terminus determines the functional specificity of KIF5A and KIF5B in dendritic spine morphogenesis.
(A) Schematic diagram of the different constructs for pull-down assay (left panel). FMRP and G3BP1 were pulled-down from the SNS by either the truncated KIF5A lacking the 88 amino acids at the …
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Figure 4—source data 1
Plotted values for Figure 4.
- https://cdn.elifesciences.org/articles/53456/elife-53456-fig4-data1-v2.xlsx
Figure 5
Mono-methylation of arginine near carboxyl-terminus of KIF5B is required for the formation of mushroom spines.
(A) Amino acid alignment of the carboxyl-termini of KIF5A, KIF5B and KIF5C. Two conserved arginine residues (R941 and R956) in KIF5B across different vertebrates were highlighted by red boxes. (B) …
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Figure 5—source data 1
Plotted values for Figure 5.
- https://cdn.elifesciences.org/articles/53456/elife-53456-fig5-data1-v2.xlsx
Figure 6 with 2 supplements
Targeting of Kif5b gene in CaMKII-Cre/Kif5bfl/fl mice and validation of the conditional knockout mice.
(A) Scheme of KIF5B knockout strategy (left panel). The blue rectangles (E1, E2, E3, E4, and E5) annotated exon 1, exon 2, exon 3, exon 4, and exon 5 of the Kif5b gene, respectively. The white …
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Figure 6—source data 1
Plotted values for Figure 6.
- https://cdn.elifesciences.org/articles/53456/elife-53456-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
KIF5B conditional knockout mice show normal general appearance and cortical layer architecture.
(A) Representative images of 7-week-old male WT and Homo mice. (B) Representative images of 7 week old WT and Homo mouse brains. Scale bar, 1 cm. (C–D) Immunohistochemical staining of NeuN-positive …
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Figure 6—figure supplement 1—source data 1
Plotted values for Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/53456/elife-53456-fig6-figsupp1-data1-v2.xlsx
Figure 6—figure supplement 2
KIF5B conditional knockout mice show no change in KIF17 expression.
Western blot analysis and quantification of KIF17 expression in total brain lysates of homozygous KIF5B conditional knockout mice (Hom) compared with control littermates (WT). n = 3 for WT. n = 3 …
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Figure 6—figure supplement 2—source data 1
Plotted values for Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/53456/elife-53456-fig6-figsupp2-data1-v2.xlsx
Figure 7 with 1 supplement
KIF5B conditional knockout mice show increase of dendritic spine instability in the frontal association cortex.
(A) Hippocampal and frontal association cortex slices of Thy1-YFP;CaMKIIα-Cre conditional Kif5bfl/fl knockout mice (Homo) and Thy1-YFP;Kif5b wild-type mice (WT) at postnatal day (P) 44 were fixed. …
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Figure 7—source data 1
Plotted values for Figure 7.
- https://cdn.elifesciences.org/articles/53456/elife-53456-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
KIF5B conditional knockout mice show no significant difference in survival rate of newly formed dendritic spines.
Survival rate of newly formed dendritic spines. The number of dendritic spines newly formed on Day 2 that persisted to Day 7 was counted as stable new spines. The stability percentage was the number …
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Figure 7—figure supplement 1—source data 1
Plotted values for Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/53456/elife-53456-fig7-figsupp1-data1-v2.xlsx
Figure 8 with 2 supplements
KIF5B conditional knockout mice show deficits in synaptic plasticity, learning and memory.
(A) Schematic diagram shows the set-up of three-chamber social interaction test and the quantification of social memory index in the right panel. n = 10 for WT. n = 11 for Hetero and Homo. Data were …
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Figure 8—source data 1
Plotted values for Figure 8.
- https://cdn.elifesciences.org/articles/53456/elife-53456-fig8-data1-v2.xlsx
Figure 8—figure supplement 1
KIF5B conditional knockout mice showed no significant abnormalities in anxiety-related behavior tests.
(A) Schematic diagram shows the experimental set-up of open field test and the quantification of time spent in center area and the total distance travelled. n = 11 for WT. n = 12 for Hetero and …
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Figure 8—figure supplement 1—source data 1
Plotted values for Figure 8—figure supplement 1.
- https://cdn.elifesciences.org/articles/53456/elife-53456-fig8-figsupp1-data1-v2.xlsx
Figure 8—figure supplement 2
Sociability, fear acquisition, and primary error of Barnes maze in KIF5B conditional knockout mice.
(A) Quantification of animal behavior in stage 1 of social interaction test. The left panel is the quantification of the total interaction time of mice with either object in stage 1 of the …
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Figure 8—figure supplement 2—source data 1
Plotted values for Figure 8—figure supplement 2.
- https://cdn.elifesciences.org/articles/53456/elife-53456-fig8-figsupp2-data1-v2.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (M. musculus) | C57/6J | The University of Hong Kong Laboratory Animal Unit | ||
| Genetic reagent (M. musculus) | Thy1-YFP-H | Jackson Laboratory. | 003782 |thy1-YFP-H | |
| Genetic reagent (M. musculus) | CaMKIIα-Cre | Jackson Laboratory. | 005359 |T29-1 | |
| Genetic reagent (M. musculus) | Kif5bfl/fl | Jiandong Huang | PMID: 20870970 | |
| Genetic reagent (M. musculus) | CaMKIIα-Cre;Kif5bfl/fl | This paper | generated from breeding of CaMKIIα-Cre and Kif5bfl/fl mice | |
| Transfected construct (M. musculus) | FLAG-677–1027 KIF5A | This paper | aa 677–1027 of mouse KIF5A with a N-terminal FLAG tag was inserted into pcDNA3 | |
| Transfected construct (M. musculus) | FLAG-677–939 KIF5A | This paper | aa 677–939 of mouse KIF5A with a N-terminal FLAG tag was inserted into pcDNA3 | |
| Transfected construct (M. musculus) | FLAG-677–938 KIF5A+941–963 KIF5B | This paper | aa 677–938 of mouse KIF5A and aa 941–963 of mouse KIF5B with a N-terminal FLAG tag was inserted into pcDNA3 | |
| Transfected construct (M. musculus) | FLAG-680–963 KIF5B | This paper | aa 680–963 of mouse KIF5B with a N-terminal FLAG tag was inserted into pcDNA3 | |
| Transfected construct (R. norvegicus) | KIF5A shRNA | This paper | 5’-TGGAAACGCCACAGATATC-3’ | |
| Transfected construct (M. musculus) | KIF5B shRNA | This paper | 5’-GGACAGATGAAGTATAAAT-3’ | |
| Transfected construct (R. norvegicus) | KIF5C shRNA | This paper | 5’-GACCCTGGCAGATGTGAAT-3’ | |
| Transfected construct (R. norvegicus) | control shRNA | Lin et al., 2017 | PMID: 28442576 | 5’-GGCTACCTCCATTTAGTGT-3’ |
| Transfected construct (M. musculus) | pKin1A | Anthony Brown | RRID:Addgene_31607 | |
| Transfected construct (M. musculus) | pcDNA3-KIF5B | Jiandong Huang | PMID: 23293293 | |
| Transfected construct (M. musculus) | pGFP-Kif5c | Michelle Peckham | RRID:Addgene_71853 | |
| Biological sample (R. norvegicus) | Primary hippocampal neuron; primary cortical neuron | Lin et al., 2017 | PMID: 28442576 | Procedures of preparing primary neurons were described in Lin et al. (2017) |
| Biological sample (M. musculus) | Synaptoneurosome | Scheetz et al., 2000 | PMID: 10700251 | Procedures of preparing synaptoneurosome were described by Scheetz et al. (2000) |
| Biological sample (M. musculus) | Synatpic plasma membrane | Bermejo et al., 2014 | PMID: 25226023 | Procedures of preparing synaptic plasma mebrane were described by Bermejo et al. (2014) |
| Antibody | KIF5B | Jiandong Huang | PMID: 20870970 | |
| Antibody | KIF5A | Abcam | RRID:AB_2132218 | |
| Antibody | KIF5C | Abcam | RRID:AB_304999 | |
| Antibody | FMRP | Abcam | RRID:AB_2278530 | |
| Antibody | KIF17 | Sigma | RRID:AB_477148 | |
| Antibody | FLAG | Sigma | RRID:AB_262044 | |
| Antibody | G3BP1 | Bethyl | RRID:AB_1576539 | |
| Antibody | G3BP2 | Bethyl | RRID:AB_1576545 | |
| Antibody | GluA2 | Millipore | RRID:AB_2113875 | |
| Antibody | RNMT | Millipore | RRID:AB_11215450 | |
| Antibody | NRGN | Millipore | Cat#AB5620 | |
| Antibody | NeuN | Millipore | Cat#AB377 | |
| Antibody | PSD-95 | NeuroMab | RRID:AB_2292909 | |
| Antibody | methylated mono-arginine R*GG | Cell Signaling | RRID:AB_10896849 | |
| Antibody | Mouse IgG2a anti-GFP | Invitrogen | RRID:AB_221568 | |
| Antibody | Rabbit anti-RFP | Rockland | RRID:AB_2209751 | |
| Antibody | Alexa 488 anti mouse IgG2a | Invitrogen | RRID:AB_2535771 | |
| Antibody | Alexa 546 anti rabbit IgG | Invitrogen | RRID:AB_2534077 | |
| Antibody | horseradish peroxidase-conjugated goat anti-rabbit IgG | Cell Signaling | RRID:AB_2099233 | |
| Antibody | horseradish peroxidase-conjugated goat anti-mouse IgG | Cell Signaling | RRID:AB_330924 | |
| Recombinant DNA reagent | pFRT-TODestFLAGHAhFMRPiso1 | Thomas Tuschl | RRID:Addgene_48690 | |
| Recombinant DNA reagent | tdTomato | Michael Davidson | RRID:Addgene_54653 | |
| Recombinant DNA reagent | GST-fused KIF5A | This paper | aa 677–1027 of mouse KIF5A was inserted into pGEX-6P-2 | |
| Recombinant DNA reagent | GST-fused KIF5B | This paper | aa 680–963 of mouse KIF5B was inserted into pGEX-6P-2 | |
| Recombinant DNA reagent | GST-fused KIF5C | This paper | aa 681–956 of mouse KIF5C was inserted into pGEX-6P-2 | |
| Recombinant DNA reagent | pEGFP-N1-KIF5A | This paper | Constructed by inserting PCR-amplified mouse KIF5A coding sequences into the pEGFP-N1 plasmid using KpnI and BamHI | |
| Recombinant DNA reagent | pEGFP-N1-KIF5B | This paper | Constructed by inserting PCR-amplified mouse KIF5B coding sequences into the pEGFP-N1 plasmid using KpnI and BamHI | |
| Sequenced-based reagent | Grin2b transcript probe (NM_012574.1, type 1) | ThermoFisher | Cat#VC1-16464 | |
| Sequenced-based reagent | Type one sense probe | ThermoFisher | Cat#VC1-20903 | |
| Sequenced-based reagent | CaMKIIa transcript probe (NM_012920.1, type 6) | ThermoFisher | Cat#VC6-11639 | |
| Sequenced-based reagent | Type six sense probe | ThermoFisher | Cat#VC6-16372 | |
| Chemical compound, drug | FLAG beads | Sigma | RRID:AB_10063035 | |
| Chemical compound, drug | glutathione sepharose four fast flow beads | GE Healthcare | Cat#17-5132-01 | |
| Chemical compound, drug | Protein A-Sepharose beads | GE Healthcare | Cat#17-5280-01 | |
| Commercial assay or kit | Lipofectamine LTX with Plus Reagent | ThermoFisher Scientific | Cat#15338100 | |
| Commercial assay or kit | SilverQuest Silver Staining Kit | Life technologies | Cat#LC6070 | |
| Commercial assay or kit | ViewRNA ISH Cell Assay Kit | ThermoFisher | Cat#QVC0001 | |
| Commercial assay or kit | Neon transfection system | ThermoFisher Scientific | Model MPK5000 | |
| Software, algorithm | Volocity | Quorum Technologies | RRID:SCR_002668 | |
| Software, algorithm | Zen digital imaging software | Zeiss | RRID:SCR_013672 | |
| Software, algorithm | Actimetrics FreezeFrame software | Coulbourn Instruments | RRID:SCR_014429 | Version 2.2 |
| Software, algorithm | ANY-maze software | ANY-maze | RRID:SCR_014289 | |
| Software, algorithm | MetaMorph software | Molecular Devices | SCR_002368 | |
| Software, algorithm | GraphPad Prism | GraphPad Prism (https://graphpad.com) | RRID:SCR_015807 | Version 6 |
| Software, algorithm | FIJI | FIJI (https://imagej.net/Fiji) | RRID:SCR_002285 | |
| Software, algorithm | KymoResliceWide | Eugene Katrukha (https://github.com/ekatrukha/KymoResliceWide) | ||
| Software, algorithm | Straighten | Eva Kocsis (https://imagej.nih.gov/ij/plugins/straighten.html) | PMID: 1817611 | |
| Software, algorithm | Mini Analysis Program | Synaptosoft | RRID:SCR_002184 | |
| Software, algorithm | CLC Main Workbench | Qiagen (https://www.qiagenbioinformatics.com/products/clc-main-workbench/) | RRID:SCR_000354 |
Additional files
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Supplementary file 1
Table for statistical tests.
- https://cdn.elifesciences.org/articles/53456/elife-53456-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/53456/elife-53456-transrepform-v2.pdf
