Local, persistent activation of Rho GTPases during plasticity of single dendritic spines

In the paper

“Amongst known effectors of Rho and Cdc42, Rock and Pak are two kinases that can be activated repsectively by these GTPases24–26.”

What the sources say

• ?Not checked — no full text of the sourcecites [24] Nikolić, M. The Pak1 kinase: an important regulator of neuronal morphology and function in the developing forebrain. Mol. Neurobiol. 37, 187–202 (2008) source
• Partly supported

  The section “R67C Mutation Impairs Kinase Activation by Cdc42” reports that PAK3 is activated by the intersectin–Cdc42 module, supporting the Pak–Cdc42 part of the claim. The passages found in our copy do not establish that ROCK is activated by Rho (searched: “ROCK” and “ROCK Rho kinase activated by Rho”). (LLM, conf=0.85)

  cites [25] Kreis, P. et al. The p21-activated kinase 3 implicated in mental retardation regulates spine morphogenesis through a Cdc42-dependent pathway. J. Biol. Chem. 282, 21497–21506 (2007) sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [26] Iden, S. & Collard, J. G. Crosstalk between small GTPases and polarity proteins in cell polarization. Nature Rev. Mol. Cell Biol. 9, 846–859 (2008) source

In the paper

“These values are ~10 times larger than the decay time constant of cytosolic paGFP (~0.4 s) and similar to that of a constitutively active HRas mutant (~6 s) (Fig. 3e–h)12.”

What the sources say

• Partly supported

  The mobility results (p. 2) report that paGFP-Ras decayed more than ten times slower than cytosolic paGFP and that constitutively active Ras G12V diffused similarly to wild-type Ras. Figure 3B’s caption (p. 8) identifies the cytosolic and Ras G12V comparisons, but our copy does not give the cited numerical estimates of ~0.4 s and ~6 s. The Rho GTPase values appear to be the citing authors’ own results, not a finding attributed to this source. (LLM, conf=0.83)

  cites [12] Harvey, C. D., Yasuda, R., Zhong, H. & Svoboda, K. The spread of Ras activity triggered by activation of a single dendritic spine. Science 321, 136–140 (2008) sourceExperimental — accuracy not yet measured

In the paper

“In contrast, HRas activity spreads from the stimulated spines along dendrites and into surrounding spines over ~10 μm12.”

What the sources say

• Supported

  The Abstract (p. 1) identifies H-Ras as the signaling molecule studied and reports that Ras activity spread approximately 10 micrometers along the dendrite and invaded neighboring spines. The spatial-profile passage (p. 2) likewise reports spread along the parent dendrite into nearby spines. (LLM, conf=0.95)

  cites [12] Harvey, C. D., Yasuda, R., Zhong, H. & Svoboda, K. The spread of Ras activity triggered by activation of a single dendritic spine. Science 321, 136–140 (2008) source

In the paper

“CaMKII activation is restricted to spines, and decays rapidly with a time constant of ~10 s13.”

What the sources say

• Partly supported

  The Discussion says CaMKII activation is largely restricted to the stimulated spine during LTP. However, the Mechanisms of spine specific CaMKII activation section reports two inactivation time constants, 6 and 45 s, rather than a single time constant of approximately 10 s. The claim captures rapid, spine-specific activation but oversimplifies its decay kinetics. (LLM, conf=0.88)

  cites [13] Lee, S. J., Escobedo-Lozoya, Y., Szatmari, E. M. & Yasuda, R. Activation of CaMKII in single dendritic spines during long-term potentiation. Nature 458, 299–304 (2009) sourceExperimental — accuracy not yet measured

In the paper

“C3 and Wasp also inhibited synaptic potentiation induced by pairing postsynaptic depolarization (0 mV) and 2-photon glutamate uncaging (Supplementary Fig. 10)13,14, suggesting that Rho and Cdc42 are important for the functional plasticity as well as the structural plasticity of spines.”

What the sources say

• Partly supported

  The section “CaMKII activation during pairing-induced LTP” (page unavailable) reports synaptic potentiation induced by pairing 0 mV postsynaptic depolarization with two-photon glutamate uncaging. Our copy does not report that C3 or Wasp inhibited this potentiation, or draw the claimed conclusion about Rho and Cdc42. (LLM, conf=0.86)

  cites [13] Lee, S. J., Escobedo-Lozoya, Y., Szatmari, E. M. & Yasuda, R. Activation of CaMKII in single dendritic spines during long-term potentiation. Nature 458, 299–304 (2009) sourceExperimental — accuracy not yet measured
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [14] Matsuzaki, M., Honkura, N., Ellis-Davies, G. C. & Kasai, H. Structural basis of long-term potentiation in single dendritic spines. Nature 429, 761–766 (2004) source

In the paper

“In contrast, inhibition of Pak with IPA3 (100 μM)28 decreased the sustained volume change selectively without changing the transient volume change (Fig. 4h, i, j) similarly to inhibition of Cdc42 signalling (Fig. 4e, g).”

What the sources say

• Supported

  The citation identifies IPA-3 as a Pak inhibitor. The source’s “IPA-3 Is a Direct, Noncompetitive Inhibitor of Pak1” section reports that IPA-3 prevents Pak1 activation; the sustained and transient volume changes are results attributed to the citing paper’s own Figure 4, not to this source. (LLM, conf=0.92)

  cites [28] Deacon, S. W. et al. An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase. Chem. Biol. 15, 322–331 (2008) source

In the paper

“The spine volume increased rapidly by ~300% following glutamate uncaging (transient phase) and relaxed to an elevated level of 70–80% for more than 30 min (sustained phase) (Figs. 1d, 2d)12–14.”

What the sources say

• Partly supported

  The paper reports that glutamate uncaging caused sustained enlargement of the stimulated spine (p. 1). Its Figure 1 caption (p. 6) describes spine-volume measurements, but the text does not give a rapid ~300% increase or a sustained 70–80% increase for more than 30 minutes; it defines the sustained measurement at 15.5–19.5 minutes. (LLM, conf=0.86)

  cites [12] Harvey, C. D., Yasuda, R., Zhong, H. & Svoboda, K. The spread of Ras activity triggered by activation of a single dendritic spine. Science 321, 136–140 (2008) sourceExperimental — accuracy not yet measured
• Partly supported

  The section “CaMKII activation during structural plasticity of dendritic spines” (page not provided) supports a rapid increase after glutamate uncaging followed by enlargement lasting more than 30 minutes. It reports 376 ± 68% initially and 104 ± 21% afterward, rather than the claimed ~300% and 70–80%. (LLM, conf=0.88)

  cites [13] Lee, S. J., Escobedo-Lozoya, Y., Szatmari, E. M. & Yasuda, R. Activation of CaMKII in single dendritic spines during long-term potentiation. Nature 458, 299–304 (2009) sourceExperimental — accuracy not yet measured
• Partly supported

  The passage labeled Abstract reports rapid spine enlargement after glutamate uncaging and enlargement lasting 70–100 minutes in 30% of stimulated spines. It gives a mean maximal volume increase of 203 ± 37%, not approximately 300%, and reports persistent increases greater than 50%, not a sustained level of 70–80%. (LLM, conf=0.88)

  cites [14] Matsuzaki, M., Honkura, N., Ellis-Davies, G. C. & Kasai, H. Structural basis of long-term potentiation in single dendritic spines. Nature 429, 761–766 (2004) sourceExperimental — accuracy not yet measured

In the paper

“Rho GTPases may constitute such signalling, because they play important roles in regulating actin cytoskeleton3,16, which is essential for spine-specific, long-term structural and functional plasticity14,17.”

What the sources say

• Supported

  The “Regulation of spine and synapse formation by small GTPases” section says that small GTPases largely regulate the actin cytoskeleton that modulates spine morphology and synapse function. The “Future directions” section likewise describes these pathways as major players in spine morphology. The sentence attaches this source’s citation to the actin-regulation clause, not the separate long-term-plasticity clause. (LLM, conf=0.93)

  cites [3] Saneyoshi, T., Fortin, D. A. & Soderling, T. R. Regulation of spine and synapse formation by activity-dependent intracellular signaling pathways. Curr. Opin. Neurobiol. 20, 108–115 (2009) source
• ?Not checked — no full text of the sourcecites [16] Heasman, S. J. & Ridley, A. J. Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nature Rev. Mol. Cell Biol. 9, 690–701 (2008) source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [14] Matsuzaki, M., Honkura, N., Ellis-Davies, G. C. & Kasai, H. Structural basis of long-term potentiation in single dendritic spines. Nature 429, 761–766 (2004) source
• ?Not checked — no full text of the sourcecites [17] Okamoto, K., Nagai, T., Miyawaki, A. & Hayashi, Y. Rapid and persistent modulation of actin dynamics regulates postsynaptic reorganization underlying bidirectional plasticity. Nature Neurosci. 7, 1104–1112 (2004) source

In the paper

“The short length constant of Cdc42 in the dendrites (1.9 μm, Fig. 3) also supports the fast inactivation of Cdc42 in the dendrite: the inactivation time constant τ is related to the length constant L and the diffusion constant D~0.6 μm2 (Ref 12) as τ = L2/D, and thus is ~6 s for Cdc42 and ~30 s for RhoA, compared to 200–300 s for HRas12.”

What the sources say

• Supported

  The cited paper identifies its Ras indicator as containing HRas (p. 1). It reports an inactivation time constant of 4.5 ± 0.4 minutes (about 270 seconds) and a diffusion coefficient of 0.65 μm²/s (p. 3), matching the approximate HRas values attributed to it. The 6-second Cdc42 and 30-second RhoA figures are calculations made by the citing authors, not findings attributed to this source. (LLM, conf=0.91)

  cites [12] Harvey, C. D., Yasuda, R., Zhong, H. & Svoboda, K. The spread of Ras activity triggered by activation of a single dendritic spine. Science 321, 136–140 (2008) source

In the paper

“In contrast, Cdc42 activation was restricted to the stimulated spine, and exhibited a steep gradient at the spine necks.”

• Also stated in: § Abstract · plain

  “In contrast, Cdc42 activation was restricted to the stimulated spines (Fig. 2a–c, 3c, d).”

In the paper

“These activities decayed over ~5 min, and were then followed by a phase of persistent activation lasting more than 30 min.”

• Also stated in: § Abstract · plain

  “The activation decayed over ~5 min, followed by sustained activity lasting more than 30 min.”

In the paper

“In contrast, shRNA against Cdc42 decreased the sustained volume change, but not the transient volume change (Fig. 4e, i, j).”

In the paper

“Downregulation of RhoA and RhoB with short-hairpin RNA (shRNA) decreased the transient volume change, but did not appreciably affect the sustained volume change (Fig. 4a, i, j).”

In the paper

“Inhibition of the Rho-Rock pathway preferentially inhibited the initial spine growth, whereas the inhibition of the Cdc42-Pak pathway blocked the maintenance of sustained structural plasticity.”

In the paper

“There was only a small fraction (10–20 %) of fluorescence remaining at 20 s after photoactivation (Fig. 3e–h), suggesting that no major immobile fraction of RhoA or Cdc42 exists in spines.”

In the paper

“The phenotypes caused by these shRNAs were rescued by co-expressing shRNA-resistant mEGFP-Rho GTPases (Fig. 4b, f, i, j), indicating that the effect of the shRNAs are specific and the mEGFP-RhoA and mEGFP-Cdc42 used in the FRET sensors are functional as endogenous proteins.”

In the paper

“Thus, our data suggest that Rho signalling is required for the transient phase and likely the sustained phase of spine enlargement, while Cdc42 signalling is required for the sustained phase.”

In the paper

“The time course of spine enlargement in neurons expressing the FRET sensor was similar to that in neurons expressing only EGFP (Fig. 4)14, suggesting that the overexpression of FRET sensors causes almost no effects on spine structural plasticity (Supplementary note).”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [14] Matsuzaki, M., Honkura, N., Ellis-Davies, G. C. & Kasai, H. Structural basis of long-term potentiation in single dendritic spines. Nature 429, 761–766 (2004) source

In the paper

“These sensors were verified to be specific and sensitive under 2pFLIM (Supplementary note).”

In the paper

“Taken together with the results from Rho/Cdc42 inhibition (Fig. 4a–j), our data implies that the Rho-Rock pathway is required for both transient and sustained phase, while the Cdc42-Pak pathway is required for the sustained phase of the structural plasticity but not for the transient phase (Fig. 4k).”

In the paper

“Since the high spatial gradient of Cdc42 between the stimulated spines and dendrite was maintained for more than 20–30 min (Fig. 2b, c, 3d), Cdc42 must be continuously activated at the stimulated spines during plasticity, and inactivated immediately after diffusing out of the spines.”

In the paper

“Thus, Cdc42 is as mobile as RhoA and HRas, yet only Cdc42 shows the compartmentalized activity.”

In the paper

“Neither C3 nor Wasp affected CaMKII activation (Supplementary Fig. 9), indicating that there is no feedback signalling from Rho and Cdc42 to upstream Ca2+ and CaMKII.”