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Cited by 206Microtubule and mitosis dynamicsCellular transport and secretionGenetic and Kidney Cyst Diseases

Stable kinesin and dynein assemblies drive the axonal transport of mammalian prion protein vesicles

Open access
Encalada, Sandra E., Szpankowski, Lukasz, Xia, Chun-hong, Goldstein, Lawrence S.B.·2011·Cell
DOI 10.1016/j.cell.2011.01.021PMC PMC3576050
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Abstract

SUMMARY Kinesin and dynein are opposite-polarity microtubule motors that drive the tightly regulated transport of a variety of cargoes. Both motors can bind to cargo but their overall composition on axonal vesicles and whether this composition directly modulates transport activity, is unknown. Here we characterize the intracellular transport and steady state motor subunit composition of mammalian prion protein (PrPC) vesicles. We identify Kinesin-1 and cytoplasmic dynein as major PrPC vesicle motor complexes, and show that their activities are tightly coupled. Regulation of normal retrograde transport by Kinesin-1 is independent of dynein-vesicle attachment, and requires the vesicle association of a complete Kinesin-1 heavy and light chain holoenzyme. Furthermore, motor subunits remain stably associated with stationary as well as with moving vesicles. Our data suggest a coordination model where PrPC vesicles maintain a stable population of associated motors whose activity is modulated by regulatory factors instead of by structural changes to motor-cargo associations.

AI snapshot

Written from the paper’s full text.

TL;DR

Mammalian prion protein (PrPC) vesicles in hippocampal axons are moved bidirectionally by Kinesin-1C/KLC1 and cytoplasmic dynein DHC1, whose activities are tightly coupled. Motor subunits remain stably associated with stationary and moving vesicles, supporting a coordination model in which activity is regulated rather than motor-cargo association.

Key findings

  • YFP-PrPC vesicles moved bidirectionally in cultured mouse hippocampal axons, with mean anterograde velocity 0.85 μm/sec (±0.036 SEM) and retrograde velocity 0.86 μm/sec (±0.06 SEM); mean run lengths were 6.2 μm (±0.5 SEM) anterograde and 4.8 μm (±0.4 SEM) retrograde, and ~70% of vesicles were stationary.
  • Kinesin-1C/KLC1 and DHC1 were identified as major PrPC vesicle motors: 58% of PrPC vesicles colocalized with KLC1 (±1.5% SEM; N=510), 35% with Kinesin-1C (±1.5% SEM; N=80), and 43% with DHC1 (±2.9% SEM; N=388); no significant colocalization was seen with Kinesin-1A.
  • Reducing Kinesin-1 subunits or DHC1 decreased run lengths, increased pause frequency, and shifted segmental velocity distributions, while mean segmental velocities were largely unchanged; Kinesin-1C removal did not dissociate DHC1 or KLC1 from PrPC vesicles.
  • Motor subunits were present on stationary as well as moving vesicles and appeared in stable, heterogeneous assemblies with quantal 1X, 2X, and 3X intensity modes, suggesting a stable motor complement whose activity is modulated by regulatory factors.

Methods

  • Live imaging of YFP-PrPC in 10-day cultured mouse hippocampal axons, with custom MATLAB/C++ particle tracking at 0.126 μm spatial and 10 Hz temporal resolution; transport parameters were compared across KLC1−/−, KLC2 shRNA, Kinesin-1A−/−, Kinesin-1C−/−, conditional Kinesin-1B cre, and DHC1 shRNA conditions, with synaptophysin-mCherry as a control cargo.
  • Biochemical vesicle immunoisolation, immunofluorescence, and a custom “motor colocalization” algorithm quantified DHC1/KLC1 association within 300 nm of PrPC vesicles; a “vesicle mapping” method in microfluidic chambers related fixed motor staining to previously recorded live vesicle trajectories.
  • Statistical analysis used nonparametric permutation t-tests and Wilcoxon-Mann-Whitney tests; velocity distributions were fit with Gaussian modes using MCLUST and Bayesian Information Criterion.

Limitations

  • Stated: whether Kinesin-1 subunits are used in different cellular contexts is unknown, and functional experiments addressing this for PrPC vesicles still need to be performed; the basis for enhanced mean velocities in Kinesin-1B-cre axons is uncertain; how Kinesin-1/dynein contacts occur is unclear.
  • Evident from the text: possible redundancy among Kinesin-1 subunits or incomplete DHC1 removal may mask effects, and the paper notes redundancy as a possible explanation for residual transport.
  • Evident from the text: motor association and copy-number estimates rely on immunofluorescence colocalization within 300 nm and intensity modes, which infer but do not directly prove physical binding; PrPC is luminal, so reverse immunoprecipitation was not possible without breaking vesicular membranes.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.cell.2011.01.021

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 7 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    YFP-PrPC vesicles reverse direction at a frequency of 0.027 switches/sec (± 0.004 switches/sec SEM).

    “The remaining vesicles reversed directions at a frequency of 0.027 switches/sec (± 0.004 switches/sec SEM).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: YFP-PrPC vesicles are representative of endogenous PrPC vesicles for the purpose of live imaging.
  • Findingown data

    Reducing Kinesin-1A and -1B slightly reduces a mode or the proportion of vesicles within higher anterograde modes.

    “Notably, reducing Kinesin-1A and -1B also slightly reduced a mode or the proportion of vesicles within higher anterograde modes (Figure 4I; Table S1)”
    Quote in the paperNot a substantial claim
  • Findingown data

    Mean retrograde segmental velocity of YFP-PrPC vesicles is 0.86 μm/sec (± 0.06 μm/sec SEM).

    “Mean anterograde and retrograde segmental velocities were 0.85 μm/sec (± 0.036 μm/sec SEM), and 0.86 μm/sec (± 0.06 μm/sec SEM; Figure 1C), respectively”
    Quote in the paperSubstantial claim
    • Rests on an assumption: YFP-PrPC vesicles are representative of endogenous PrPC vesicles for the purpose of live imaging.
  • Findingown data

    PrPC and KHC immunoprecipitate with KLC1 in floated membrane fractions.

    “Using an antibody against KLC1 to pull-down associated membrane components, we found that PrPC and KHC (as detected by an antibody that recognizes primarily Kinesin-1C) immunoprecipitated with KLC1, as did the amyloid precursor protein (APP)”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The Kinesin-1C antibody used recognizes primarily Kinesin-1C.
  • Findingown data

    YFP-PrPC vesicles move bidirectionally in wild-type hippocampal axons.

    “In wild-type neurons, YFP-PrPC vesicles moved in anterograde and retrograde directions, and a large percentage were stationary”
    Quote in the paperSubstantial claim
    • Rests on an assumption: YFP-PrPC vesicles are representative of endogenous PrPC vesicles for the purpose of live imaging.
  • May combine two claims. 34
  • Classified as probably not a substantial claim. 2

  • May combine two claims.

    Findingown data

    Reduction of DHC1 by shRNA disrupts bidirectional YFP-PrPC vesicle transport, decreasing run lengths and increasing the frequency of pauses.

    “reduction of DHC1 (mRNA reduced by 80-90%, protein reduced by 66%; Figures S2A, S2B, S5D, and S5E), disrupted bidirectional YFP-PrPC vesicle transport, decreasing run lengths and increasing the frequency of pauses”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The DHC1 shRNA specifically reduces DHC1 without off-target effects on transport.

  • May combine two claims.

    Findingown data

    In wild-type axons, three and two modes best fit the anterograde and retrograde YFP-PrPC velocity distributions, respectively.

    “In wildtype, three and two modes best fit the anterograde and retrograde distributions, respectively (Figures 2F, 3F, and 4I; Table 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Kinesin-1C -/- axons, the proportion of anterograde-moving PrPC vesicles declines, run lengths in both directions are significantly decreased, and vesicles pause more frequently in both directions.

    “In Kinesin-1C -/- axons, the proportion of anterograde moving PrPC vesicles declined, run lengths in both directions were significantly decreased, and these vesicles paused more frequently when moving in both directions (Figures 4A-C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Kinesin-1B-cre axons, anterograde movement is either unchanged or activated, as demonstrated by longer runs and faster mean segmental velocities.

    “However, anterograde movement was either unchanged (pause frequencies; Figure 4G), or activated as demonstrated by longer runs, and strikingly faster mean segmental velocities (Figures 4F and 4H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Wild-type anterograde and retrograde segmental velocity distributions of YFP-PrPC vesicles are non-normal and right-skewed.

    “Wild-type anterograde and retrograde segmental velocity distributions were non-normal and showed a right-skewed bias (Figure 1D).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    No major changes in YFP-PrPC transport are observed in Kinesin-1A -/- axons, except a minor decrease in retrograde run lengths and slight changes in segmental velocity distributions.

    “We did not observe major changes in YFP-PrPC transport in Kinesin-1A -/- axons, as only a minor decrease in retrograde run lengths and slight changes in segmental velocity distributions were observed (Figures 4A-D, and 4I; see next section).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In wild-type axons, 43% of PrPC vesicles colocalize with DHC1 (± 2.9% SEM), 57% associate with KLC1 (± 1.5% SEM), 25% have both motor subunits (± 1.5% SEM), and 23% have no motor subunits (± 1.8% SEM).

    “We found that in wild-type axons, 43% of PrPC vesicles colocalized with DHC1 (± 2.9% SEM), 57% of PrPC vesicles associated with KLC1 (± 1.5% SEM), and 25% of PrPC vesicles had both motor subunits (± 1.5% SEM). We did not detect motor subunits on 23% of PrPC vesicles (± 1.8% SEM).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Motor puncta detected within 300 nm of a PrPC vesicle are associated with that vesicle.

  • May combine two claims.

    Findingown data

    Reducing KLC1 or KLC2 function causes a significantly decreased percentage of anterograde-moving YFP-PrPC vesicles and a higher frequency of stalled particles.

    “Reducing the function of each KLC subunit caused a significantly decreased percentage of anterograde-moving vesicles, and a higher frequency of stalled particles (Figures 3A and 3B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mean segmental velocities of YFP-PrPC vesicles are largely unaffected in KLC mutants, with the exception of slight increases in KLC1-/- neurons.

    “Given the pronounced reductions of bidirectional movement, mean segmental velocities were surprisingly largely unaffected in KLC mutants, with the exception of slight increases in KLC1 -/- neurons contributed solely by the small number of reversing vesicles (Figures S3A-C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Decreasing DHC1 results in bidirectional shifts from higher to lower velocity modes and in a decreased number of modes.

    “Decreasing DHC1 also resulted in bidirectional shifts from higher to lower velocity modes and in a decreased number of modes (Figure 2F; Table S1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Combining velocity distributions after reducing each KLC partially reconstitutes the wild-type anterograde distribution but not the retrograde one.

    “We tested this by combining velocity distributions after reducing each KLC, and observing the partial reconstitution of the wild-type anterograde distribution but not the retrograde one (Figure 3G; Table S1).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Genetic removal of Kinesin-1C or other motor subunits does not cause compensatory changes that confound the measured motor subunit distributions.

  • May combine two claims.

    Findingown data

    Disrupting either KLC1 or Kinesin-1C resulted in almost identical bidirectional phenotypes, more severe than seen with other subunits.

    “disrupting either of these motor subunits resulted in almost identical bidirectional phenotypes, more severe than seen with other subunits, including the decrease in bidirectional velocity distributions (Table S1).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Genetic removal of Kinesin-1C or other motor subunits does not cause compensatory changes that confound the measured motor subunit distributions.

  • May combine two claims.

    Findingown data

    KLC1 remains vesicle associated in Kinesin-1C mutants, so its presence alone is not sufficient to activate normal retrograde motion.

    “However, KLC1 remains vesicle associated in Kinesin-1C mutants, so its presence alone is not sufficient to activate normal retrograde motion.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Genetic removal of Kinesin-1C or other motor subunits does not cause compensatory changes that confound the measured motor subunit distributions.

  • May combine two claims.

    Findingown data

    Coordination of retrograde activity by Kinesin-1C is independent of DHC1-vesicle association but involves the simultaneous attachment of both types of motors to vesicles.

    “Our data show that coordination of retrograde activity by Kinesin-1C is independent of DHC1-vesicle association, but involves the simultaneous attachment of both types of motors to vesicles (Figures 5B).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: YFP-PrPC vesicles are representative of endogenous PrPC vesicles in axons.
    • Rests on an assumption: Antibodies against KLC1 and DHC1 specifically and quantitatively detect these motor subunits on vesicles.

  • May combine two claims.

    Findingown data

    Impairing Kinesin-1 and DHC1 resulted in PrPC vesicles that traveled shorter distances, paused more frequently, and had altered velocity distributions.

    “Activity changes were revealed after impairing Kinesin-1 and DHC1, which resulted in PrPC vesicles that traveled shorter distances, paused more frequently, and had altered velocity distributions (Figures 3, 4).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: YFP-PrPC vesicles are representative of endogenous PrPC vesicles in axons.
    • Rests on an assumption: Antibodies against KLC1 and DHC1 specifically and quantitatively detect these motor subunits on vesicles.

  • May combine two claims.

    Interpretationown data

    Velocity distributions are reduced as a result of removal or decreases of opposite polarity motors, suggesting that regulation of motors can contribute to these activity changes.

    “and that velocity distributions are reduced as a result of removal or decreases of opposite polarity motors, suggesting that regulation of motors can contribute to these activity changes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Kinesin-1A and -1B are not major components of the PrPC vesicular transport machinery.

    “These results suggest that Kinesin-1A, and -1B are not major components of the PrPC vesicular transport machinery”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    PrPC vesicles move bidirectionally en route to the synapse in primary hippocampal neurons with dynamics consistent with MT-dependent fast axonal transport mediated by kinesin and dynein motor proteins.

    “Thus, vesicles containing YFP-PrPC move bidirectionally en route to the synapse in primary hippocampal neurons with dynamics that are consistent with MT-dependent fast axonal transport mediated by kinesin and dynein motor proteins.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The presence of KLC1 on a PrPC vesicle does not influence the association of DHC1, and vice versa.

    “No changes to these distributions were observed when both KLC1 and DHC1 were associated simultaneously with vesicles or when Kinesin-1C was removed, suggesting that presence of KLC1 on the vesicle did not influence the association of DHC1 and vice versa”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Fluorescence intensity of motor subunit staining on a vesicle is proportional to the number of motor subunits, so discrete intensity modes correspond to 1X, 2X, and 3X motor numbers.

  • May combine two claims.

    Interpretationown data

    KLC1 and KLC2 mediate anterograde transport of YFP-PrPC vesicles and also activate retrograde motility.

    “Thus, KLC1 and KLC2 mediate anterograde transport of YFP-PrPC vesicles and also activate retrograde motility.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    PrPC moves in anterograde and retrograde directions in nerves.

    “Previous studies showed that PrPC moved in anterograde and retrograde directions in nerves”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Borchelt et al., 1994 source

      Not checked in the free run.

    • cites Butowt et al., 2006 source

      Not checked in the free run.

    • cites Moya et al., 2004 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    KLC1 is necessary but not sufficient to activate normal retrograde transport of PrPC vesicles.

    “we conclude that KLC1 is necessary but not sufficient to activate normal retrograde transport of PrPC vesicles.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Kinesin-1C and DHC1 are the major anterograde and retrograde motors required to transport PrPC vesicles in mammalian axons.

    “We identified Kinesin-1C and DHC1 as major anterograde and retrograde motors required to transport PrPC vesicles in mammalian axons”
    Quote in the paperSubstantial claim
    • Rests on an assumption: YFP-PrPC vesicles are representative of endogenous PrPC vesicles in axons.
    • Rests on an assumption: Antibodies against KLC1 and DHC1 specifically and quantitatively detect these motor subunits on vesicles.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Kinesin-1C mRNA expression is upregulated in Kinesin-1B null extraembryonic membranes.

    “Kinesin-1C mRNA expression was upregulated in Kinesin-1B null extraembryonic membranes”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Tanaka et al., 1998 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Motor subunit association is necessary but not sufficient for active translocation of PrPC vesicles along microtubules.

    “indicating that motor subunit association is necessary but not sufficient for active translocation along microtubules.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Fixation and immunostaining preserve the motor subunit composition on PrPC vesicles as it was during live imaging, allowing fixed vesicles to be mapped to their live trajectories.
    • Rests on an assumption: YFP-PrPC vesicles are representative of endogenous PrPC vesicles in axons.
    • Rests on an assumption: Antibodies against KLC1 and DHC1 specifically and quantitatively detect these motor subunits on vesicles.

  • May combine two claims.

    Interpretationown data

    The incomplete disruption of anterograde transport after reduction of any one KHC suggests either redundancy or the requirement of another unidentified motor.

    “suggesting either some redundancy, or the requirement of another as yet unidentified motor.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Genetic removal of Kinesin-1C or other motor subunits does not cause compensatory changes that confound the measured motor subunit distributions.

  • May combine two claims.

    Interpretationown data

    KLC1, Kinesin-1C, and DHC1, but not Kinesin-1A, associate with PrPC vesicles in vivo.

    “Thus, KLC1, Kinesin-1C, and DHC1, but not Kinesin-1A, associate with PrPC vesicles in vivo.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Motor puncta detected within 300 nm of a PrPC vesicle are associated with that vesicle.
    • Rests on an assumption: The Kinesin-1C antibody used recognizes primarily Kinesin-1C.

  • May combine two claims.

    Interpretationown data

    Kinesin-1C and DHC1 are the main plus- and minus-end motors for PrPC vesicle transport, respectively, with a minor role for Kinesin-1A and Kinesin-1B.

    “identified Kinesin-1C and DHC1 as the main plus- and minus-end motors, respectively, with a minor role attributed to Kinesin-1A and Kinesin-1B.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: YFP-PrPC vesicles are representative of endogenous PrPC vesicles in axons.
    • Rests on an assumption: Antibodies against KLC1 and DHC1 specifically and quantitatively detect these motor subunits on vesicles.

  • May combine two claims.

    Interpretationown data

    Kinesin-1C is not required for DHC1 or KLC1 association with PrPC vesicles.

    “We conclude that Kinesin-1C is not required for DHC1 or KLC1 association with PrPC vesicles.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Motor puncta detected within 300 nm of a PrPC vesicle are associated with that vesicle.

  • May combine two claims.

    Interpretationreasoning

    The data are consistent with a model for bidirectional coordination in which activities of Kinesin-1 and dynein might be linked via physical contacts, and point to a role of KLCs in this linkage.

    “Thus, our data is consistent with a model for bidirectional coordination in which activities of Kinesin-1 and dynein might be linked via physical contacts (Martin et al., 1999), and points to a role of KLCs in this linkage.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Martin et al., 1999 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The requirements for KLC1, KLC2, Kinesin-1C and DHC1 in PrPC vesicle transport are specific, as movement of synaptophysin vesicles is not inhibited following their disruption.

    “These requirements appear specific, as movement of synaptophysin vesicles is not inhibited following their disruption.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: YFP-PrPC vesicles are representative of endogenous PrPC vesicles in axons.
    • Rests on an assumption: Antibodies against KLC1 and DHC1 specifically and quantitatively detect these motor subunits on vesicles.

  • May combine two claims.

    Interpretationreasoning

    A stable population of Kinesin-1 subunits and DHC1 associate with PrPC vesicles, and a subset of these activate bidirectional movement while the rest remain vesicle-bound but inactive.

    “We thus propose a coordination model in which a stable population of Kinesin-1 subunits and DHC1 associate with PrPC vesicles and a subset of these activate bidirectional movement, while the rest remain vesicle-bound but inactive (Figure 7A).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: YFP-PrPC vesicles are representative of endogenous PrPC vesicles in axons.
    • Rests on an assumption: Antibodies against KLC1 and DHC1 specifically and quantitatively detect these motor subunits on vesicles.

  • May combine two claims.

    Interpretationown data

    Kinesin-1C performs a dual function as a mediator of anterograde movement and an activator of retrograde transport, but is not required for cargo binding by dynein.

    “Kinesin-1C thus appears to perform a dual function as a mediator of anterograde movement, and as an activator of retrograde transport, but is not required for cargo binding by dynein.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: YFP-PrPC vesicles are representative of endogenous PrPC vesicles in axons.
    • Rests on an assumption: Antibodies against KLC1 and DHC1 specifically and quantitatively detect these motor subunits on vesicles.

  • May combine two claims.

    Interpretationown data

    Retrograde activation occurs by the formation and vesicle association of an intact Kinesin-1C/KLC1 complex, which is required for proper retrograde activity.

    “Our data suggest that it does so by the formation and vesicle association of an intact Kinesin-1C/KLC1 complex, which is required for proper retrograde activity.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: YFP-PrPC vesicles are representative of endogenous PrPC vesicles in axons.
    • Rests on an assumption: Antibodies against KLC1 and DHC1 specifically and quantitatively detect these motor subunits on vesicles.

  • May combine two claims.

    Interpretationown data

    The data support regulatory motor coordination and argue against simple tug-of-war scenarios.

    “thus providing support for regulatory motor coordination, and against simple TOW scenarios.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: YFP-PrPC vesicles are representative of endogenous PrPC vesicles in axons.
    • Rests on an assumption: Antibodies against KLC1 and DHC1 specifically and quantitatively detect these motor subunits on vesicles.

  • May combine two claims.

    Premisecitation

    Kinesin-1C is decreased but still present in KLC1 -/- cells, suggesting that this KHC subunit alone cannot induce normal retrograde activity.

    “Likewise, Kinesin-1C is decreased but still present in KLC1 -/- cells (Rhiannon Killian, personal communication), suggesting that this KHC subunit alone cannot induce normal retrograde activity.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Rhiannon Killian, personal communication

      Not checked in the free run.

36 of 36

  • The paper relies on this without support.

    YFP-PrPC vesicles are representative of endogenous PrPC vesicles for the purpose of live imaging.

    “we tracked individual moving vesicles in 10-day old cultured mouse hippocampal axons from neurons transfected with a YFP-PrPC fusion construct”

  • The paper relies on this without support.

    Motor puncta detected within 300 nm of a PrPC vesicle are associated with that vesicle.

    “A custom-built ‘motor colocalization’ algorithm quantified presence or absence and intensity amplitudes of each detected DHC1 and/or KLC1 puncta within 300nm of each PrPC vesicle”

  • The paper relies on this without support.

    The Kinesin-1C antibody used recognizes primarily Kinesin-1C.

    “as detected by an antibody that recognizes primarily Kinesin-1C”

  • The paper relies on this without support.

    The DHC1 shRNA specifically reduces DHC1 without off-target effects on transport.

    “reduction of DHC1 (mRNA reduced by 80-90%, protein reduced by 66%; Figures S2A, S2B, S5D, and S5E), disrupted bidirectional YFP-PrPC vesicle transport”

  • The paper relies on this without support.

    Fluorescence intensity of motor subunit staining on a vesicle is proportional to the number of motor subunits, so discrete intensity modes correspond to 1X, 2X, and 3X motor numbers.

    “we divided PrPC vesicles into those associated with a single motor subunit (either KLC1 or DHC1), or with both, and quantified their intensity distributions and the percentage of vesicles within each predicted mode.”

  • The paper relies on this without support.

    Fixation and immunostaining preserve the motor subunit composition on PrPC vesicles as it was during live imaging, allowing fixed vesicles to be mapped to their live trajectories.

    “Movement of vesicles was imaged and cells were fixed with paraformaldehyde for subsequent staining with KLC1 and DHC1 antibodies, but the trajectories of vesicles pre- and post-fixation were recorded (Figure 6B).”

  • The paper relies on this without support.

    YFP-PrPC vesicles are representative of endogenous PrPC vesicles in axons.

    “Hippocampal cells were plated in microfluidic chambers to promote the growth of straight axons, and were transfected with YFP-PrPC (Taylor et al., 2005; Figure 6A).”

  • The paper relies on this without support.

    Antibodies against KLC1 and DHC1 specifically and quantitatively detect these motor subunits on vesicles.

    “stained with antibodies against KLC1 and DHC1.”

  • The paper relies on this without support.

    Genetic removal of Kinesin-1C or other motor subunits does not cause compensatory changes that confound the measured motor subunit distributions.

    “No changes to these distributions were observed when both KLC1 and DHC1 were associated simultaneously with vesicles or when Kinesin-1C was removed”
9 of 9

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Studies have shown cargoes that maintain a stable population of associated motors when changing direction or pausing, suggesting regulation of motor activity rather than relative changes in motor association.

    “Recently, however, studies have shown cargoes that maintain a stable population of associated motors when changing direction, or even pausing, suggesting regulation of motor activity rather than relative changes in motor association (Encalada et al., 2011).”

    From Microtubule motors transport phagosomes in the RPE, and lack of KLC1 leads to AMD-like pathogenesis (2015) · cites this paper as Encalada et al., 2011

  • Studies of axonal transport of vesicles containing prion protein have shown that KLC1 is required for normal retrograde transport activity.

    “Studies of the axonal transport of vesicles containing prion protein have shown that KLC1 is also required for normal retrograde transport activity (Encalada et al., 2011).”

    From Microtubule motors transport phagosomes in the RPE, and lack of KLC1 leads to AMD-like pathogenesis (2015) · cites this paper as Encalada et al., 2011

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Vesicle-association percentages do not reconcile

    Minor

    The reported DHC1-positive and KLC1-positive percentages, with 25% positive for both, imply that 25% have neither motor subunit. The paper instead reports 23% with neither, leaving the four stated vesicle categories totaling 98% rather than 100%.

1 of 1

The citation lists haven’t been fetched yet

References 59 · Cited by 206

Research works printed but not linked: 13

  1. Cui, 2010, Targeted inactivation of kinesin-1 in pancreatic beta-cells in vivo leads to insulin secretory deficiency, Diabetes
  2. Baas, P.W., Deitch, J.S., Black, M.M., and Banker, G.A. (1988). Polarity orientation of microtubules in hippocampal neurons: uniformity in the axon and nonuniformity in the dendrite. Proc. Natl. Acad. Sci. USA 85, 8335–8339. · DOI · Paper page
  3. Borchelt, D.R., Davis, J., Fischer, M., Lee, M.K., Slunt, H.H., Ratovitsky, T., Regard, J., Copeland, N.G., Jenkins, N.A., Sisodia, S.S., et al. (1996). A vector for expressing foreign genes in the brains and hearts of transgenic mice. Genet. Anal. 13, 159–163. · DOI
  4. Falzone, T.L., Stokin, G.B., Lillo, C., Rodrigues, E.M., Westerman, E.L., Williams, D.S., and Goldstein, L.S. (2009). Axonal stress kinase activation and tau misbehavior induced by kinesin-1 transport defects. J. Neurosci. 29, 5758–5767. · DOI
  5. Fraley, C. (1999). MCLUST: Software for Model-Based Cluster Analysis. Journal of Classification 16, 297–306. · DOI
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  7. Jaqaman, K., Loerke, D., Mettlen, M., Kuwata, H., Grinstein, S., Schmid, S.L., and Danuser, G. (2008). Robust single-particle tracking in live-cell time-lapse sequences. Nat. Methods 5, 695–702. · DOI
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Authors
Encalada, S. E., Szpankowski, L., Xia, C.-H., & Goldstein, L. S. B.
Affiliations
  • University of California San Diego (US)
  • Howard Hughes Medical Institute (US)
Corresponding author
  • Encalada, Sandra E.
  • Goldstein, Lawrence S.B.
ORCID
Venue
Cell
Publisher
Elsevier BV
Published
2011-02
Volume
144
Issue
4
Pages
551-565
ISSN
0092-8674 (ISSN-L)
DOI
10.1016/j.cell.2011.01.021
PMC
PMC3576050
OpenAlex
W2048921995
Semantic Scholar
6099e349adf6c8535d5b603e7f3e6b026381364e
Cited by
206
References
59
Citations by year
2026: 7 · 2025: 10 · 2024: 12 · 2023: 16 · 2022: 13 · 2021: 16 · 2020: 20 · 2019: 16 · 2018: 16 · 2017: 14 · 2016: 6 · 2015: 7 · 2014: 17 · 2013: 12 · 2012: 15
FWCI
8.07
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institute on Aging (AG000216, R01AG032180, AG032180, T32AG000216)
  • National Institute of General Medical Sciences (T32GM008806)
  • National Institute of Neurological Disorders and Stroke (P30NS047101)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic and Kidney Cyst Diseases
Keywords
prion protein, axonal transport, kinesin-1, cytoplasmic dynein, retrograde transport, microtubule motor proteins
MeSH terms
Animals, Axons, Dyneins, Hippocampus, Mice, Inbred C57BL, Motor Activity, Neurons, Kinesins, PrPC Proteins, Transport Vesicles, Mice

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