TRIM3 Regulates the Motility of the Kinesin Motor Protein KIF21B

<div><p>Kinesin superfamily proteins (KIFs) are molecular motors that transport cellular cargo along the microtubule cytoskeleton. KIF21B is a neuronal kinesin that is highly enriched in dendrites. The regulation and specificity of microtubule transport involves the binding of motors to individual cargo adapters and accessory proteins. Moreover, posttranslational modifications of either the motor protein, their cargos or tubulin regulate motility, cargo recognition and the binding or unloading of cargos. Here we show that the ubiquitin E3 ligase TRIM3, also known as BERP, interacts with KIF21B via its RBCC domain. TRIM3 is found at intracellular and Golgi-derived vesicles and co-localizes with the KIF21B motor in neurons. <i>Trim3</i> gene deletion in mice and TRIM3 overexpression in cultured neurons both suggested that the E3-ligase function of TRIM3 is not involved in KIF21B degradation, however TRIM3 depletion reduces the motility of the motor. Together, our data suggest that TRIM3 is a regulator in the modulation of KIF21B motor function.</p> </div

The E3 ligase TRIM3 is not involved in the degradation of the motor protein KIF21B.

<p>(A, B) KIF21B half life analysis using cycloheximide (CHX) chase experiments. More than 50% of KIF21B degrades within 48 hours in cultured hippocampal neurons (DIV16) derived from wildtype (+/+) mice. TRIM3 genetic depletion does not alter the half life of KIF21B, as assessed through evaluation of relative KIF21B signal intensities. Optineurin and actin served as controls. Relative signal intensity of KIF21B/actin ratios in %. n=3, each. 4h: wildtype (+/+) 100%, knockout (-/-) 100%; 8h: wildtype (+/+) 93.9±9.2, knockout (-/-) 110.4±12.9; 24h: wildtype (+/+) 57.8±11.5, knockout (-/-) 61.5±12.0; 48h: wildtype (+/+) 39.4±10.3, knockout (-/-) 35.4±9.6. (C, D) Relative signal intensities of KIF21B and KIF5 in hippocampal lysates remain equal across the genotypes (wildtype (+/+) versus <i>Trim3</i> knockout (-/-)). Relative signal intensity of KIF/NSE ratios in %. n=4 each. KIF21B: wildtype (+/+) 0.55±0.08, knockout (-/-) 0.69±0.03; KIF5: wildtype (+/+) 0.70±0.07, knockout (-/-): 0.72±0.03. ns: not significant. (E, F) Overexpression of TRIM3 does not alter endogenous KIF21B protein levels. Cultured hippocampal neurons (DIV10) were transfected with vectors encoding HA-TRIM3 or HA, respectively. Coexpression of GFP served as transfection control and volume marker. Cells were fixed and stained for endogenous KIF21B at DIV14. Somatic KIF21B signal intensity: HA-control: set to 100%, n= 40; HA-TRIM3: 104±17%, n=38. (Scale bars in E: 20 µm.) Data: means±SEM.</p

Subcellular localization of KIF21B and TRIM3.

<p>(A) KIF21B locates to the somato-dendritic compartment of neurons and is prominent in growth cones of young neurons (DIV7) (arrows). (Scale bar: 20 µm.) (B) Electron microscopy analysis, immunogold signals. TRIM3 locates close to the membrane of putative transport vesicles in neurons derived from hippocampal slices (arrow) (Scale bar: 50 nm.). (C) Electron microscopy analysis, DAB signals. TRIM3 locates to vesicles at the Golgi apparatus (arrows). M: mitochondria, Golgi: Golgi apparatus (Scale bar: 200 nm). (D, E) TRIM3 (red, left) or KIF21B (red, right) colocalize with MAP2-positive dendrites (green, arrows) but are not detected in GFAP-positive astrocytes (blue, arrowheads). (Scale bars: 20 µm.) (F) KIF21B and TRIM3 colocalize in punctate structures (yellow, arrowheads) across neuronal dendrites. (Scale bar: 20 µm, scale bar boxed region: 5 µm.).</p

Interaction of KIF21B and TRIM3 in vitro.

<p>(A) Co-immunoprecipitation: a KIF21B-specific antibody precipitates endogenous KIF21B and co-precipitates endogenous TRIM3 from brain lysate indicating <i>in </i><i>vitro</i> binding of both proteins. (B) Schematic representation of the domain structures of KIF21B and TRIM3. WD40-repeats: tryptophan-aspartic acid (W-D) dipeptide repeats; R: RING; B:B-box; CC: Coiled-coil; ABP: ABP (actin-binding protein)-like domain; NHL: NCL-1/HT2A/Lin-41. (C) Mapping of interaction domains using the DupLEX-A yeast two-hybrid-system. Full-length TRIM3 and the TRIM3-RBCC-domain (aa1-290) bind the stalk region of the motor protein KIF21B. Beta-galactosidase activity (blue signals). Values represent average signal intensities (arbitrary units).</p

Analysis of mCherry-KIF21B mobility in wildtype (+/+) and <i>Trim3</i> knockout (-/-) neurons.

<p>(A-E) Cultured hippocampal neurons at DIV6-11. (A, B) Particles in wildtype (+/+) neurons were analysed with an image acquisition rate: 1 image/sec. The corresponding kymograph represents a region of 80µm. (C) Schematic representation of mobile (arrows in A) and stationary particles. (D) Quantitative evaluation of instantaneous particle speed across the genotypes (+/+: 1.47+0.20 µm/sec; -/-: 0.44+0.22 µm/sec). (E) Quantitative evaluation of the number of stationary particles across the genotypes (+/+: 60.90+9.76%; -/-: 91.40+3.90%). (F-I) FRAP-analysis of mCherry-KIF21B in cultured hippocampal neurons (DIV11) derived from wildtype (+/+) or <i>Trim3</i> knockout (-/-) mice. After photobleaching of transfected cultured neurons, the recovery of mCherry-KIF21B fluorescent signals was subsequently analysed over 500 sec with an image acquisition rate of 1 image/5 sec. mCherry was analysed with an image acquisition rate of 1 image/sec (control). (F, G) mCherry-KIF21B in wildtype (+/+) and in <i>Trim3</i> knockout (-/-) neurons. Scale bars: 10 µm. Kymographs display the bleached regions and the recovery of fluorescence over 6 min. (H) Quantification of mCherry-KIF21B fluorescent recovery in wildtype (+/+) and <i>Trim3</i> knockout (-/-) neurons. The recovery rate is much faster in wildtype (+/+) neurons, as compared to <i>Trim3</i> knockout (-/-) neurons. (I) Control: quantification of mCherry fluorescent recovery in wildtype (+/+) and <i>Trim3</i> knockout (-/-) neurons reveals equal values. (J, K) Analysis of KIF21B content in pellet and supernatant fractions by Triton-X-100 extraction. Pellet (P) and supernatant (S) fractions derived from cultured hippocampal wildtype (+/+) or <i>Trim3</i> knockout (-/-) neurons (DIV20-23) were subjected to Western blot analysis. Quantification of KIF21B content in supernatant: wildtype (+/+) values set to 1 (3 individual experiments with a total of n=10), <i>Trim3</i> knockout (-/-)= 0.65±0.08 (3 individual experiments with a total of n=8). (a.u.= arbitrary units.) Data: means±SEM.</p

Generation of a <i>Trim3</i> knockout mouse and validation of TRIM3 depletion.

<p>(A) Schematic representation of the <i>Trim3</i> gene and the <i>Trim3</i> targeting construct. Homologous recombination of the targeting construct produces a mutant <i>Trim3</i> gene containing a Neo^r cassette flanked by frt sites (grey boxes, f) and loxP sites (white boxes, l) flanking exons 3-5. Recombination of the loxP sites results in excision of exons 3-5; recombination of the frt sites results in excision of the Neo^r cassette. Forward primers (a-d), reverse primers (1-4) and BglII restriction sites (▲) used for genotyping are indicated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075603#pone.0075603.s001" target="_blank">Figure S1C</a>). (B) Western blotting confirms the absence of TRIM3 in hippocampus (Hc), cerebellum (Cb) and cortex (Cx). (C) Immunocytochemistry confirms the absence of TRIM3 in cultured hippocampal neurons derived from <i>Trim3</i> knockout mice (DIV12). Control: Synaptic vesicle protein SV2. (Scale bars: 20 µm.).</p