A live-cell marker to visualize the dynamics of stable microtubules throughout the cell cycle

May combine two claims.

In vitro, at high concentrations in the no-nucleotide state, the kinesin-1 motor domain can extend microtubule lattice spacing, suggesting it prefers to bind to the expanded lattice conformation at lower concentrations.

“Third, in vitro experiments have revealed that at high concentrations in the no-nucleotide state, the kinesin-1 motor domain can extend MT lattice spacing, suggesting that it prefers to bind to this expanded MT lattice conformation at lower concentrations (Peet et al., 2018; Shima et al., 2018).”

• cites Peet et al., 2018 sourceSupported
• cites Shima et al., 2018 sourceSupported

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

Stable microtubules have a curved appearance.

“A characteristic feature of stable MTs is their curved appearance (Piperno et al., 1987; Friedman et al., 2010; Katrukha et al., 2017).”

• cites Piperno et al., 1987 sourcePartly supported

  In the Results (Fig. 1), the paper says that some acetylated microtubules in HeLa cells are curved. Its drug-resistance results associate acetylated microtubules with greater stability, but it does not establish curvature as a characteristic feature of stable microtubules generally. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites Friedman et al., 2010 sourceSupported
• cites Katrukha et al., 2017 sourcePartly supported

  In Results, ‘Kinesin-1-targeted microtubules display undulations’ (Fig. 3e), the paper reports that the acetylated microtubules preferred by kinesin-1 are highly curved and describes them as a subset of stable microtubules. It does not establish curved appearance as a characteristic of stable microtubules generally. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

Microtubule-binding proteins can induce artifacts of the microtubule cytoskeleton when highly overexpressed, as observed with the N-terminal fragment of Ensconsin/MAP7.

“A potential limitation of our approach is that MT-binding proteins can induce artifacts of the MT cytoskeleton when highly overexpressed, as has, for example, been observed with the N-terminal fragment of Ensconsin/MAP7 (Faire et al., 1999).”

• cites Faire et al., 1999 sourceSupported

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

The presence of the N-terminal FKBP domain in the Rab6a construct has no detectable effects on the behavior of this marker.

“The presence of the N-terminal FKBP domain in the Rab6a construct has no detectable effects on the behavior of this marker (Schlager et al., 2014; Serra-Marques et al., 2020).”

• cites Schlager et al., 2014 sourceCould not verify

  Figure 1 and the section on kinesin-driven Rab6 movement show that the authors used and imaged FKBP-tagged Rab6 vesicles. Our copy does not report a comparison with Rab6 lacking FKBP or state that the N-terminal FKBP domain has no detectable effect on marker behavior (searched for FKBP-tag effects and tagged-versus-untagged controls). (LLM, conf=0.72)

  Experimental — accuracy not yet measured
• cites Serra-Marques et al., 2020 sourceSupported

May combine two claims.

Increasing MT detyrosination by VSH1/SVBP overexpression does not confer stability to microtubules; the majority of detyrosinated MTs are lost upon nocodazole treatment.

“we found that increasing MT detyrosination levels by overexpression of VSH1/SVBP does not confer stability to MTs as the majority of detyrosinated MTs were lost upon treatment with nocodazole (Fig. S1 G), in agreement with earlier work (Khawaja et al., 1988).”

• cites Khawaja et al., 1988 sourceSupported

Classified as probably not a substantial claim.

Earlier work has described increased lifetime of stable microtubules compared with dynamic microtubules.

“Earlier work has described the increased lifetime of stable MTs compared with dynamic MTs (Schulze and Kirschner 1986; Webster et al., 1987; Infante et al., 2000).”

• cites Schulze and Kirschner 1986 sourceSupported
• cites Webster et al., 1987 sourceSupported
• cites Infante et al., 2000 sourceNot checked — no full text of the source

May combine two claims. The quote only partly states this claim.

Stable microtubules are not static; they display sliding at approximately 0.7 μm/s, active curvature induction, deformation, breaking, and occasional depolymerization.

“We found that, although these MTs were stable, they were far from static and displayed distinct behaviors such as sliding (at ∼0.7 µm/s, Fig. 3, C and F; Video 4), active curvature induction (Fig. 3 E; Video 5), deformation (Fig. S3 A), and breaking (Fig. S3 B), consistent with earlier reports (Xu et al., 2017; Robison et al., 2016; Katrukha et al., 2017).”

• Also stated in: § Results · plain

  “Occasionally, (partial) depolymerization events of stable MTs were observed (Fig. S3 C).”

• cites Xu et al., 2017Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Robison et al., 2016 sourcePartly supported

  In “MTs buckle under contractile load” (page unavailable), Robison et al. report that microtubules deform and buckle during cardiomyocyte contraction, supporting the cited comparison that stable microtubules need not be static. The Discussion (page unavailable) says they normally buckle rather than break or slide; the paper does not report sliding at ∼0.7 µm/s, active curvature induction, or occasional depolymerization. Its sliding-anchor mechanism is a model, not an observation of microtubule sliding. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Katrukha et al., 2017 sourcePartly supported

  In Results, under “Kinesin-1-targeted microtubules display undulations” (page unavailable), the paper reports that a subset of stable microtubules changed shape over time through undulation-like motion. The Discussion also describes active shape remodelling. Our copy does not report sliding at approximately 0.7 µm/s, breaking, or occasional microtubule depolymerization; its mention of depolymerization concerns actin. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

May combine two claims.

The G234A rigor mutant of Kinesin-1 binds to and effectively decorates acetylated microtubules.

“Similar to the active motor, the G234A rigor mutant of Kinesin-1 binds to and effectively decorates acetylated MTs (Tas et al., 2017; Guardia et al., 2016; Farías et al., 2017).”

• cites Tas et al., 2017 sourcePartly supported

  The Results section (page unavailable) reports that a Kif5a rigor mutant binds microtubules and shows dramatic colocalization with acetylated tubulin, supporting the binding and decoration claim. Our copy does not identify that mutant as G234A, so it does not verify the specific mutation attributed to this source. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites Guardia et al., 2016 sourceSupported
• cites Farías et al., 2017 sourcePartly supported

  The Results (Fig. 5F; page not provided) report that a kinesin-1 rigor mutant binds to and decorates microtubule tracks in the initial axon. The paper identifies the mutant as G235A, not G234A, and does not identify those tracks as acetylated microtubules. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

May combine two claims.

Distribution of mitochondria throughout the cell is Kinesin-1-dependent, and absence of Kinesin-1 or its activating MAP, MAP7, leads to clustering of mitochondria around the nucleus.

“Distribution of mitochondria throughout the cell is Kinesin-1–dependent and the absence of Kinesin-1 or its activating MAP, MAP7, leads to the clustering of mitochondria around the nucleus.”

• cites Hooikaas et al., 2019 sourcePartly supported

  The Results section reports that loss of the kinesin-1 motor KIF5B caused mitochondria to cluster around the nucleus in HeLa cells. It also says that loss of MAP7 alone caused no mitochondrial-localization defect; clustering occurred when MAP7, MAP7D1, and MAP7D3 were depleted together. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites Serra-Marques et al., 2020 sourcePartly supported

  The Results section ('KIF5B and KIF13B are the main drivers of Rab6 vesicle transport in HeLa cells'; page unavailable) reports that mitochondria shifted toward the perinuclear region in cell lines lacking KIF5B. The Discussion says MAP7 family members recruit and activate KIF5B, but our copy does not show that loss of MAP7 causes mitochondrial clustering. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Rab6a secretory vesicles are transported from the Golgi to the plasma membrane by the concerted action of Kif5b (Kinesin-1) and Kif13b (Kinesin-3).

“Rab6a secretory vesicles are transported from the Golgi to the plasma membrane by the concerted action of Kif5b (Kinesin-1) and Kif13b (Kinesin-3; Serra-Marques et al., 2020).”

• cites Serra-Marques et al., 2020 sourceSupported

May combine two claims.

Dynamic microtubules can be readily visualized in live cells using plus-end markers or a sensor for tyrosinated tubulin, whereas stable microtubules cannot currently be visualized live except upon cold treatment, microtubule-depolymerizing drugs, or immunocytochemistry for posttranslational modifications.

“Dynamic MTs can be readily visualized in live cells using plus-end markers or the recently developed sensor for tyrosinated tubulin (Kesarwani et al., 2020); however, this is not the case for stable MTs, which can currently only be visualized upon cold treatment or exposure to MT-depolymerizing drugs, or approximated using immunocytochemistry for different PTMs.”

• cites Kesarwani et al., 2020 sourceSupported

May combine two claims.

StableMARK molecules quickly detach from dynamic microtubules while remaining stably bound to long-lived microtubules.

“However, StableMARK molecules quickly detach from dynamic MTs while remaining stably bound to long-lived MTs.”

May combine two claims. The quote only partly states this claim.

Nocodazole induces rapid loss of most mCherry-tubulin-labeled microtubules, whereas StableMARK-decorated microtubules remain largely unaffected; after 30 minutes a clear network of StableMARK-positive microtubules is still observed.

“The addition of 10 µM nocodazole induced a rapid loss of most MTs labeled with mCherry-tubulin, whereas the subset of StableMARK-decorated MTs remained largely unaffected by the treatment.”

• Also stated in: § Results · plain

  “Indeed, 30 min after the addition of nocodazole, a clear network of StableMARK-positive MTs could still be observed (Fig. S1 H).”

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

Release of the midbody could sometimes be observed.

“Eventually, abscission of the cytokinetic bridge took place, and sometimes the release of the midbody could be observed (Fig. S6 E).”

May combine two claims.

Initially, the developing spindles were StableMARK-negative, and StableMARK-positive microtubules appeared fragmented.

“Initially, the developing spindles were StableMARK-negative, and the StableMARK-positive MTs appeared fragmented (Fig. 8 A).”

The quote only partly states this claim.

Rab6a vesicles are transported along StableMARK-decorated microtubules.

“we could directly observe the transport of Rab6a vesicles along StableMARK-decorated MTs (Fig. 5, C–E; Video 6).”

May combine two claims.

Low StableMARK expression does not affect the density of EB1 comets or the microtubule growth rate.

“in low-StableMARK-expressing cells (i.e., at concentrations below 2 μM), the density of EB1 comets was similar to control cells, indicating that the amount of polymerizing MTs in the cell is not affected by low levels of StableMARK (Fig. 4, C and D).”

• Also stated in: § Results · plain

  “we assessed the MT growth rate in control and low-StableMARK-expressing cells using the growing plus-end marker EB3-tdTomato and found no difference between the two conditions (Fig. 4, E and F).”

May combine two claims.

In low StableMARK-expressing cells, mitochondria were spread throughout the cytoplasm in most cells (66%), similar to control cells (70%), whereas high-expressing cells had clustered mitochondria (67%).

“While in high-expressing cells, mitochondria were tightly clustered around the nucleus in the majority of cells (67%), in low-expressing cells, mitochondria were spread throughout the cytoplasm in most cells (66%), similar to what is seen for control cells (70%; Fig. 5, A and B).”

May combine two claims.

StableMARK-decorated microtubules remain stable on the imaging timescale of several minutes, whereas many microtubules labeled only by mCherry-tubulin display dynamic instability.

“we observed that many MTs labeled exclusively by mCherry-tubulin displayed dynamic instability, whereas MTs decorated by StableMARK remained stable on the imaging timescale of several minutes (Fig. 3 A; Video 2).”

May combine two claims.

Taxol promotes the binding of StableMARK to all microtubules in cells and increases its binding to microtubules in vitro.

“Consistently, Taxol, an MT stabilizer that induces a small longitudinal expansion of the MT lattice (Estévez-Gallego et al., 2020; Rai et al., 2020; Alushin et al., 2014; Vale et al., 1994), promotes the binding of StableMARK to all MTs in cells and increases its binding to MTs in vitro.”

May combine two claims.

At high StableMARK expression, there is a correlation between StableMARK expression and MT acetylation levels, and high expression induces bundling of acetylated microtubules.

“At high expression levels, we observed a clear correlation between StableMARK expression and MT acetylation levels, with high StableMARK-expressing cells having increased levels of MT acetylation (Fig. 4 B and Fig. S4 B).”

• Also stated in: § Results · plain

  “In addition, high StableMARK expression induced bundling of acetylated MTs (Fig. 4 A).”

May combine two claims. The quote only partly states this claim.

StableMARK-decorated microtubules respond to serum starvation and serum readdition similarly to control stable microtubules and are not overstabilized.

“This indicates that the subset of stable MTs can still undergo changes and respond to physiological cues in presence of StableMARK. In other words, StableMARK-decorated MTs are not overstabilized as they behave similarly to non-StableMARK-decorated (i.e., in non-expressing cells) stable MTs during prolonged serum starvation.”

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

Normality was assumed, not formally tested, for the ratio paired t test of the means.

“*P < 0.05 (ratio paired t test of the means; normality assumed, not formally tested).”

Classified as probably not a substantial claim. May combine two claims.

EB proteins bind to the tips of polymerizing microtubules and are often used to visualize the subset of dynamic microtubules.

“EB proteins bind to the tips of polymerizing MTs and are often used to visualize the subset of dynamic MTs (Akhmanova and Steinmetz 2015).”

• cites Akhmanova and Steinmetz 2015 sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Counting molecules by eye from timelapse acquisition and sum projections ensured no molecules were overlooked.

“Subsequently, the number of molecules along the traced MTs was counted by eye based on the timelapse acquisition of the molecules (61 frames) and sum projections thereof to ensure no molecules were overlooked.”

May combine two claims.

StableMARK labels stable, long-lived microtubules in cells, many of which are nocodazole resistant.

“Instead, it appears to recognize a specific expanded lattice conformation, allowing it to label stable, long-lived MTs in cells, many of which are nocodazole resistant.”

May combine two claims.

StableMARK-decorated long-lived microtubules have varying degrees of lattice stabilization, with about half not depolymerizing upon laser-induced severing.

“These results indicate that StableMARK-decorated MTs have varying degrees of stability.”

• Also stated in: § Discussion · plain

  “our work reveals that long-lived MTs have varying degrees of lattice stabilization, with about half of them not depolymerizing upon laser-induced severing.”

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

Illumination settings were kept the same for all images acquired from a particular coverslip.

“Illumination settings were kept the same for all images acquired from a particular coverslip.”

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

For the acetylation, EB1, and cytochrome C control experiments, transfected and non-transfected cells from the same coverslip were randomly selected for imaging.

“For the acetylation, EB1, and cytochrome C control experiments, transfected and non-transfected cells from the same coverslip were randomly selected for imaging.”

May combine two claims.

StableMARK molecules randomly sample all microtubules in the cell, but only become locked in their high-affinity state when encountering a stable microtubule.

“Based on this data, we propose that StableMARK molecules randomly sample all MTs in the cell, but only become locked in their high-affinity state when encountering a stable MT (Fig. 7 G).”

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

During classification of mitochondria distribution, the observer was unaware of the transfection status of the classified cell.

“During the classification of mitochondria distribution, the observer was unaware of the transfection status of the classified cell.”

Classified as probably not a substantial claim.

To quantify the effect of StableMARK expression on MT acetylation levels, a large number of StableMARK- and non-expressing cells were randomly imaged to capture the expression range of StableMARK under similar conditions.

“To quantify the effect of StableMARK expression on MT acetylation levels, we randomly imaged a large number of StableMARK- and non-expressing cells to capture the expression range of StableMARK in any given experiment performed under similar conditions.”

May combine two claims.

The analyzed MTs did not depolymerize during StableMARK imaging, as verified by comparing MT images before and after imaging.

“This was compared for MT images before and after imaging StableMARK to ensure the MT did not depolymerize.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Stable microtubules are characterized by long lifetime and resistance to microtubule-depolymerizing drugs, but the exact origin of their stability has remained largely unknown.

“Stable MTs are characterized by their long lifetime and resistance to MT-depolymerizing drugs, yet the exact origin of their stability has remained largely unknown (Akhmanova and Kapitein 2022).”

• cites Akhmanova and Kapitein 2022 sourceNot checked — no full text of the source

May combine two claims.

Stable microtubules are enriched in posttranslational modifications such as acetylation and detyrosination, and immunolabeling for these modifications is often used as a proxy for stable microtubules.

“Stable MTs are enriched in PTMs like acetylation and detyrosination, and immunolabeling for these modifications is often used as a proxy for stable MTs in experimental work.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Cells were confirmed to be free of mycoplasma.

“Cells were confirmed to be free of mycoplasma.”

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

Stabilization of long-lived microtubules starts by stabilization of the plus-end or other sites on the lattice, followed by stabilization of the microtubule along the whole lattice, for example via lattice expansion.

“This could reflect an aging effect in which stabilization starts by stabilization of the plus-end (or other sites on the lattice), followed by stabilization of the MT along the whole lattice, e.g., via lattice expansion.”

May combine two claims.

Stabilization starts by modifying the microtubule plus-end to create a long-lived microtubule, followed by gradual stabilization of the rest of the lattice, for example by accumulation of MAPs and/or PTMs, which might eventually alter the lattice and induce lattice expansion.

“This variation among StableMARK-decorated MTs suggests that stabilization starts by modifying the MT plus-end to create a long-lived MT, followed by gradual stabilization of the rest of the lattice, for example, by the accumulation of MAPs and/or PTMs, which might eventually also alter the lattice of the MT, inducing lattice expansion.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Illumination settings were chosen so that low-StableMARK-expressing cells could be detected while no pixel saturation occurred for high-expressing cells.

“Illumination settings were chosen so that low-StableMARK-expressing cells could be detected, while at the same time, no pixel saturation occurred for high-expressing cells.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

For the colocalization coefficients of acetylated tubulin to StableMARK across conditions, data did not pass the Shapiro-Wilk normality test, so Kruskal-Wallis with Dunn's multiple comparisons was used.

“Data were checked for normality by Shapiro–Wilk test. Because data did not pass the normality test, Kruskal–Wallis test with Dunn’s multiple comparisons test was used to compare the colocalization coefficients of acetylated tubulin to StableMARK for the different conditions.”

Classified as probably not a substantial claim.

All in vitro assays were performed paired to ensure that pipetting errors or other variability could not account for observed differences.

“Importantly, all assays were performed paired to ensure that pipetting errors or any other variability could not account for any observed differences (Fig. S2 B).”

May combine two claims.

StableMARK is a live-cell marker for stable microtubules that allows direct visualization of the dynamics of stable microtubules.

“Here, we introduced StableMARK as a live-cell marker for stable MTs that allows for the direct visualization of the dynamics of stable MTs.”

May combine two claims.

The G234A point mutation in Kinesin-1 perturbs its ATPase activity, resulting in a motor protein with a very low rate of microtubule unbinding.

“The G234A point mutation in Kinesin-1 perturbs its ATPase activity (Rice et al., 1999), resulting in a motor protein that has a very low rate of MT unbinding.”

• cites Rice et al., 1999 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Because these experiments are sensitive to sample preparation and microscope performance, results of an independent duplicate experiment are reported separately in the supplemental materials.

“As these types of experiments are very sensitive to variations in sample preparation and microscope performance, results of an independent duplicate experiment are reported separately in the supplemental materials.”

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

The construct was verified by sequencing.

“The construct was verified by sequencing.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

For the fluorescence intensity data comparing drug treatments to the DMSO control, data did not pass the Shapiro-Wilk normality test, so Kruskal-Wallis with Dunn's multiple comparisons was used.

“Data were checked for normality by Shapiro–Wilk test. Because data did not pass the normality test, Kruskal–Wallis test with Dunn’s multiple comparisons test was used to compare the different conditions to the DMSO control.”

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

For all other fixed experiments, illumination settings were kept similar for all conditions.

“For all other fixed experiments, illumination settings were kept similar for all conditions.”

May combine two claims.

The surface of stable microtubules is different from the surface of dynamic microtubules, and this differentiation is recognized by StableMARK and prompts them to adopt a high microtubule-affinity state.

“These findings suggest that the surface of stable MTs is different from the surface of dynamic MTs and that this differentiation of the MT surface is recognized by StableMARK and prompts them to adopt a high MT-affinity state.”

May combine two claims.

The FCS setup was validated by measuring a dilution series of fluorescein sodium salt in PBS, and it could reliably measure concentrations between 10 nM and 100 μM.

“We validated the dynamic range of our FCS setup by measuring a dilution series of fluorescein sodium salt (518-47-8; Sigma-Aldrich) in PBS and concluded that we could reliably measure concentrations ranging between 10 nM and 100 µM.”

May combine two claims.

The off-rate of StableMARK is approximately zero in all conditions tested in vitro, and this could limit the difference observed.

“Note that the off-rate of StableMARK is ∼0 in all conditions tested in vitro and this could limit the difference observed.”