Specific depletion of the motor protein KIF5B leads to deficits in dendritic transport, synaptic plasticity and memory

  1. Junjun Zhao
  2. Albert Hiu Ka Fok
  3. Ruolin Fan
  4. Pui-Yi Kwan
  5. Hei-Lok Chan
  6. Louisa Hoi-Ying Lo
  7. Ying-Shing Chan
  8. Wing-Ho Yung
  9. Jiandong Huang
  10. Cora Sau Wan Lai  Is a corresponding author
  11. Kwok-On Lai  Is a corresponding author
  1. The University of Hong Kong, China
  2. Chinese University of Hong Kong, China
  3. Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, China
8 figures, 1 table and 2 additional files

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 …

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 …

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 …

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 …

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 …

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;…

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 …

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) …

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 …

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 …

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 …

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. …

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 …

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 …

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 …

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 …

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (M. musculus)C57/6JThe University of Hong Kong Laboratory Animal Unit
Genetic reagent (M. musculus)Thy1-YFP-HJackson Laboratory.003782 |thy1-YFP-H
Genetic reagent (M. musculus)CaMKIIα-CreJackson Laboratory.005359 |T29-1
Genetic reagent (M. musculus)Kif5bfl/flJiandong HuangPMID: 20870970
Genetic reagent (M. musculus)CaMKIIα-Cre;Kif5bfl/flThis papergenerated from breeding of CaMKIIα-Cre and Kif5bfl/fl mice
Transfected construct (M. musculus)FLAG-677–1027 KIF5AThis paperaa 677–1027 of mouse KIF5A with a N-terminal FLAG tag was inserted into pcDNA3
Transfected construct (M. musculus)FLAG-677–939 KIF5AThis paperaa 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 KIF5BThis paperaa 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 KIF5BThis paperaa 680–963 of mouse KIF5B with a N-terminal FLAG tag was inserted into pcDNA3
Transfected construct (R. norvegicus)KIF5A shRNAThis paper5’-TGGAAACGCCACAGATATC-3’
Transfected construct (M. musculus)KIF5B shRNAThis paper5’-GGACAGATGAAGTATAAAT-3’
Transfected construct (R. norvegicus)KIF5C shRNAThis paper5’-GACCCTGGCAGATGTGAAT-3’
Transfected construct (R. norvegicus)control shRNALin et al., 2017PMID: 284425765’-GGCTACCTCCATTTAGTGT-3’
Transfected construct (M. musculus)pKin1AAnthony BrownRRID:Addgene_31607
Transfected construct (M. musculus)pcDNA3-KIF5BJiandong HuangPMID: 23293293
Transfected construct (M. musculus)pGFP-Kif5cMichelle PeckhamRRID:Addgene_71853
Biological sample (R. norvegicus)Primary hippocampal neuron; primary cortical neuronLin et al., 2017PMID: 28442576Procedures of preparing primary neurons were described in Lin et al. (2017)
Biological sample (M. musculus)SynaptoneurosomeScheetz et al., 2000PMID: 10700251Procedures of preparing synaptoneurosome were described by Scheetz et al. (2000)
Biological sample (M. musculus)Synatpic plasma membraneBermejo et al., 2014PMID: 25226023Procedures of preparing synaptic plasma mebrane were described by Bermejo et al. (2014)
AntibodyKIF5BJiandong HuangPMID: 20870970
AntibodyKIF5AAbcamRRID:AB_2132218
AntibodyKIF5CAbcamRRID:AB_304999
AntibodyFMRPAbcamRRID:AB_2278530
AntibodyKIF17SigmaRRID:AB_477148
AntibodyFLAGSigmaRRID:AB_262044
AntibodyG3BP1BethylRRID:AB_1576539
AntibodyG3BP2BethylRRID:AB_1576545
AntibodyGluA2MilliporeRRID:AB_2113875
AntibodyRNMTMilliporeRRID:AB_11215450
AntibodyNRGNMilliporeCat#AB5620
AntibodyNeuNMilliporeCat#AB377
AntibodyPSD-95NeuroMabRRID:AB_2292909
Antibodymethylated mono-arginine R*GGCell SignalingRRID:AB_10896849
AntibodyMouse IgG2a anti-GFPInvitrogenRRID:AB_221568
AntibodyRabbit anti-RFPRocklandRRID:AB_2209751
AntibodyAlexa 488 anti mouse IgG2aInvitrogenRRID:AB_2535771
AntibodyAlexa 546 anti rabbit IgGInvitrogenRRID:AB_2534077
Antibodyhorseradish peroxidase-conjugated goat anti-rabbit IgGCell SignalingRRID:AB_2099233
Antibodyhorseradish peroxidase-conjugated goat anti-mouse IgGCell SignalingRRID:AB_330924
Recombinant DNA reagentpFRT-TODestFLAGHAhFMRPiso1Thomas TuschlRRID:Addgene_48690
Recombinant DNA reagenttdTomatoMichael DavidsonRRID:Addgene_54653
Recombinant DNA reagentGST-fused KIF5AThis paperaa 677–1027 of mouse KIF5A was inserted into pGEX-6P-2
Recombinant DNA reagentGST-fused KIF5BThis paperaa 680–963 of mouse KIF5B was inserted into pGEX-6P-2
Recombinant DNA reagentGST-fused KIF5CThis paperaa 681–956 of mouse KIF5C was inserted into pGEX-6P-2
Recombinant DNA reagentpEGFP-N1-KIF5AThis paperConstructed by inserting PCR-amplified mouse KIF5A coding sequences into the pEGFP-N1 plasmid using KpnI and BamHI
Recombinant DNA reagentpEGFP-N1-KIF5BThis paperConstructed by inserting PCR-amplified mouse KIF5B coding sequences into the pEGFP-N1 plasmid using KpnI and BamHI
Sequenced-based reagentGrin2b transcript probe (NM_012574.1, type 1)ThermoFisherCat#VC1-16464
Sequenced-based reagentType one sense probeThermoFisherCat#VC1-20903
Sequenced-based reagentCaMKIIa transcript probe (NM_012920.1, type 6)ThermoFisherCat#VC6-11639
Sequenced-based reagentType six sense probeThermoFisherCat#VC6-16372
Chemical compound, drugFLAG beadsSigmaRRID:AB_10063035
Chemical compound, drugglutathione sepharose four fast flow beadsGE HealthcareCat#17-5132-01
Chemical compound, drugProtein A-Sepharose beadsGE HealthcareCat#17-5280-01
Commercial assay or kitLipofectamine LTX with Plus ReagentThermoFisher ScientificCat#15338100
Commercial assay or kitSilverQuest Silver Staining KitLife technologiesCat#LC6070
Commercial assay or kitViewRNA ISH Cell Assay KitThermoFisherCat#QVC0001
Commercial assay or kitNeon transfection systemThermoFisher ScientificModel MPK5000
Software, algorithmVolocityQuorum TechnologiesRRID:SCR_002668
Software, algorithmZen digital imaging softwareZeissRRID:SCR_013672
Software, algorithmActimetrics FreezeFrame softwareCoulbourn InstrumentsRRID:SCR_014429Version 2.2
Software, algorithmANY-maze softwareANY-mazeRRID:SCR_014289
Software, algorithmMetaMorph softwareMolecular DevicesSCR_002368
Software, algorithmGraphPad PrismGraphPad Prism (https://graphpad.com)RRID:SCR_015807Version 6
Software, algorithmFIJIFIJI (https://imagej.net/Fiji)RRID:SCR_002285
Software, algorithmKymoResliceWideEugene Katrukha (https://github.com/ekatrukha/KymoResliceWide)
Software, algorithmStraightenEva Kocsis (https://imagej.nih.gov/ij/plugins/straighten.html)PMID: 1817611
Software, algorithmMini Analysis ProgramSynaptosoftRRID:SCR_002184
Software, algorithmCLC Main WorkbenchQiagen (https://www.qiagenbioinformatics.com/products/clc-main-workbench/)RRID:SCR_000354

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