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. 2024 Jan 12;35(2):ar23. doi: 10.1091/mbc.E23-09-0370

MARCKS and PI(4,5)P2 reciprocally regulate actin-based dendritic spine morphology

Barbara Calabrese a,*, Shelley Halpain a,*
Editor: Stephanie Guptonb
PMCID: PMC10881156  PMID: 38088877

Abstract

Myristoylated, alanine-rich C-kinase substrate (MARCKS) is an F-actin and phospholipid binding protein implicated in numerous cellular activities, including the regulation of morphology in neuronal dendrites and dendritic spines. MARCKS contains a lysine-rich effector domain that mediates its binding to plasma membrane phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) in a manner controlled by PKC and calcium/calmodulin. In neurons, manipulations of MARCKS concentration and membrane targeting strongly affect the numbers, shapes, and F-actin properties of dendritic spines, but the mechanisms remain unclear. Here, we tested the hypothesis that the effects of MARCKS on dendritic spine morphology are due to its capacity to regulate the availability of plasma membrane PI(4,5)P2. We observed that the concentration of free PI(4,5)P2 on the dendritic plasma membrane was inversely proportional to the concentration of MARCKS. Endogenous PI(4,5)P2 levels were increased or decreased, respectively, by acutely overexpressing either phosphatidylinositol-4-phosphate 5-kinase (PIP5K) or inositol polyphosphate 5-phosphatase (5ptase). PIP5K, like MARCKS depletion, induced severe spine shrinkage; 5ptase, like constitutively membrane-bound MARCKS, induced aberrant spine elongation. These phenotypes involved changes in actin properties driven by the F-actin severing protein cofilin. Collectively, these findings support a model in which neuronal activity regulates actin-dependent spine morphology through antagonistic interactions of MARCKS and PI(4,5)P2.

  • Dendritic spines are essential for brain plasticity. Disruptions in their number and size are linked to neurological disorders. MARCKS and cofilin are known dendritic spine regulators that interact with PI(4,5)P2 How all three cooperate to control spines is unknown.

  • Using high-resolution imaging, the authors found that membrane-bound MARCKS interferes with PI(4,5)P2-mediated cofilin inhibition, resulting in elongated spines; loss of MARCKS shrinks spines via boosted cofilin inhibition by PI(4,5)P2.

  • This study shows how three regulators of actin dynamics, MARCKS, PI(4,5)P2 and cofilin, coordinately regulate spine morphology. This is broadly relevant to actin-membrane interactions in many tissues, and specifically to synapse structure in health and disease.

INTRODUCTION

Regulation of the plasma membrane and its underlying actin cytoskeleton are essential in shaping the dynamic morphological properties of cells, including the formation of dendritic spines along dendrites, which are the postsynaptic portion of excitatory glutamatergic synapses (Calabrese et al., 2006; Patterson and Yasuda, 2011; Penzes and Rafalovich, 2012; Okabe, 2020). Structural plasticity of spines accompanies changes in synaptic strength during long-term depression (LTD) and long-term potentiation (LTP), two forms of neuronal network plasticity that underlie learning (Patterson and Yasuda, 2011; Bosch and Hayashi, 2012).

Myristoylated, alanine-rich C-kinase substrate (MARCKS) is a protein well-positioned to regulate such structural plasticity and spine development due to its direct binding to filamentous actin (F-actin) and to the acidic phospholipid phosphatidylinositol-4,5-biphosphate (PI(4,5)P2), which, as one of its predominant functions, signals to the actin cytoskeleton (El Amri et al., 2018; Mandal, 2020; Chen et al., 2021). Biophysical studies in cell-free systems demonstrated that the polybasic effector domain of MARCKS shows high affinity and selectivity for PI(4,5)P2 (Wang et al., 2002; McLaughlin and Murray, 2005). This domain contains 12 lysines within its 25 amino acids, and each effector domain reportedly binds up to three PI(4,5)P2 molecules, thereby “sequestering” PI(4,5)P2 and preventing its interaction with its effectors (McLaughlin and Aderem, 1995; McLaughlin et al., 2002; Rauch et al., 2002; Gambhir et al., 2004). Like MARCKS, PI(4,5)P2 has significant roles in regulating many aspects of neuronal function (El Amri et al., 2018; Katan and Cockcroft, 2020; Mandal, 2020). MARCKS is enriched in the brain relative to other tissues (Albert et al., 1986; Blackshear et al., 1986; Ouimet et al., 1990), where it is a major substrate of protein kinase C (PKC) (Albert et al., 1986; Ouimet et al., 1990). It regulates multiple aspects of neuronal function, including the differentiation of neural precursors, dendrite arborization, and axonal regeneration (Saito and Shirai, 2002; Sun and Alkon, 2014; Callender and Newton, 2017; Geribaldi-Doldan et al., 2019). Phosphorylation by PKC within the MARCKS effector domain disrupts its electrostatic binding to PI(4,5)P2, thereby inhibiting MARCKS interaction with the plasma membrane (Hartwig et al., 1992; Kim et al., 1994). Ca2+/calmodulin binding to the effector domain has a similar effect (Arbuzova et al., 1997). Thus, MARCKS regulates PI(4,5)P2 availability in response to changes in intracellular calcium.

Previously we reported that endogenous MARCKS is present in dendritic spines and strongly regulates their morphology (Calabrese and Halpain, 2005). Spines display a variable level of MARCKS, (Ouimet et al., 1990; Calabrese and Halpain, 2005). Using cultured hippocampal neurons, we found that MARCKS silencing using shRNA induced a sizeable decrease in spine numbers and shrinkage of those remaining. On the other hand, overexpressing wildtype MARCKS induced aberrantly elongated dendritic spines. Constitutively driving more MARCKS onto the membrane, via ectopic expression of mutated MARCKS that is nonphosphorylatable at four key serine residues within the effector domain (S4N-MARCKS), had even stronger effects. Both these manipulations (i.e., both reduced and excessive MARCKS at the membrane) caused significant reductions in spine numbers, but did so via different mechanisms, with spine loss from decreased MARCKS attributed to extensive spine shrinkage, and spine loss from increased membrane MARCKS attributed to aberrant membrane dynamics causing spines to fuse (Calabrese and Halpain, 2005).

In the present study we tested the hypothesis that these dramatic changes in dendritic spine morphology are due to the propensity of MARCKS to reduce the availability of PI(4,5)P2 (Rauch et al., 2002; Gambhir et al., 2004). Our results show that, consistent with this hypothesis, MARCKS is an important regulator of PI(4,5)P2 on dendrite and dendritic spine membrane, and that reciprocal manipulations of MARCKS and PI(4,5)P2 have remarkably similar effects on spine morphology. Moreover, we find that the effects of both MARCKS and PI(4,5)P2 are influenced by the activity of the actin severing and dynamizing protein cofilin.

RESULTS

PI(4,5)P2 distribution in the postsynaptic compartment

To evaluate the concentration and distribution of endogenous PI(4,5)P2 along the dendrites in cultured hippocampal neurons, we expressed a red fluorescently tagged pleckstrin homology domain (PH) of phospholipase C (PLC) δ1 (mRFP-PHPLCδ1; Balla et al., 2000). When expressed at low levels, this PH domain binds with high affinity to PI(4,5)P2 without altering cell physiology (Halet, 2005). Consistent with previous reports (Czech, 2000; van Rheenen et al., 2007; Balla and Varnai, 2009; Al-Fahad et al., 2022), mRFP-PHPLCδ1 was predominantly localized along the plasma membrane (Figure 1), a localization that is especially evident in optical sections through the cell body (Figure 1A, inset). The mRFP-PHPLCδ1 signal was also notably enriched in the majority of dendritic spines relative to the adjacent dendritic shaft (Figure 1, A and B), and strongly colocalized with the specific postsynaptic protein PSD-95. Quantitative analyses showed that 68.3 ± 3.7% of spines were positive for both PHPLCδ1 and PSD-95. On average across dendritic regions 36.6 ± 2.8% of pixels that were positive for PSD-95 were also positive for PHPLCδ1, and 33.8 ± 2.8% of pixels that were positive for PHPLCδ1 were also positive for PSD-95. These data confirm prior reports that PI(4,5)P2 is enriched at postsynaptic sites (Horne and Dell’Acqua, 2007; Hofbrucker-MacKenzie et al., 2023).

FIGURE 1:

FIGURE 1:

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PI(4,5)P2 localization in dissociated hippocampal neurons is redistributed in response to MARCKS. (A) Representative neuron coexpressing soluble eGFP with the PI(4,5)P2 reporter mRFP- mRFP-PHPLCδ1 (here shortened to “mRFP-PH,”) which is largely enriched at the plasma membrane (see inset: cell body) and present in the majority of dendritic spines (red arrow). Scale bar, 10 µm. (B) Two representative examples of dendritic spines coexpressing mRFP-PH and PSD95-GFP. Most of the mRFP-PH enriched areas partially overlap with PSD-95 clusters (red arrows). Scale bars (for both examples): 2 µm. (C) Hippocampal dendritic regions of neurons coexpressing mRFP-PH with either eGFP, eGFP-S4N-MARCKS, or MARCKS shRNA (see insets). All image widths = 14 µm. (D) Fluorescence intensity (FI) of the mRFP-PH probe relative to the representative purple line scan across the dendrites shown in (C). (E) “Membrane association index” (MAI) for mRFP-PH in neurons expressing either eGFP, S4N-MARCKS or MARCKS shRNA (see Materials and Methods on how MAI was calculated). Data are expressed as mean ± SEM; *** p < 0.001, **** p < 0.0001 for representative comparisons. The full set of comparisons, test selection, and statistical data are provided in Supplemental Table S1.

MARCKS controls PI(4,5)P2 availability in hippocampal neurons

The mRFP-PHPLCδ1 probe can be thought of as a quantitative reporter of the availability of “free” PI(4,5)P2 (i.e., PI(4,5)P2 that is neither sequestered by MARCKS, or other MARCKS-like polybasic proteins, nor bound by PI(4,5)P2 effectors). We therefore used the probe to test whether increasing or decreasing the concentration of MARCKS on the plasma membrane would exhibit the postulated effect of decreasing or increasing, respectively, free PI(4,5)P2. First, we confirmed this prediction qualitatively in COS-7 cells (Supplemental Figure S1). We then devised a quantitative assay for free PI(4,5)P2 in neurons using line scans across spine-free stretches of dendrite where we could reliably calculate a “membrane association index (MAI)” (see Materials and Methods) that reflects the concentration of free PI(4,5)P2 at the plasma membrane relative to the presumptive unbound mRFP-PHPLCδ1 probe, observed as lower fluorescence intensity within the cytoplasm (Figure 1, C–E). We confirmed that plasma membrane-association of the probe is acutely regulated by neuronal stimuli that affect PI(4,5)P2 metabolism and availability. Brief incubation of cultures with 40 µM N-methyl d-aspartate (NMDA) induced a transient increase in free PI(4,5)P2 on the dendrite membrane, followed by a delayed decrease (Supplemental Figure S2; Supplemental Table S1). Although more difficult to quantify, free PI(4,5)P2 levels in dendritic spines appeared to follow a similar pattern. Our observations are consistent with a recent report that used immunogold labeling and electron microscopy to discover that spine plasma membrane PI(4,5)P2 transiently increases during brief exposure to NMDA (Hofbrucker-MacKenzie et al., 2023).

Relative to eGFP-transfected controls, neurons coexpressing constitutively membrane-bound MARCKS-S4N showed a 40% decrease in the membrane association of mRFP-PHPLCδ1 (Figure 1, D and E; Supplemental Table S1). In neurons where shRNA was used to deplete endogenous MARCKS, there was an 18.4% increase in the membrane association of mRFP-PHPLCδ1. These results demonstrate the antagonistic effect of membrane-bound MARCKS on the availability of free PI(4,5)P2 in neurons, consistent with prior studies using nonneuronal cells and cell-free biophysical approaches (Glaser et al., 1996; Laux et al., 2000; McLaughlin et al., 2002; Dietrich et al., 2009; Yamaguchi et al., 2009; Jahan et al., 2020).

PI(4,5)P2 mediates the effects of MARCKS on dendritic spine morphology

We next addressed the hypothesis that MARCKS regulates dendritic spine morphology by controlling PI(4,5)P2 availability. We predicted that a reduction in endogenous PI(4,5)P2 should mimic the phenotypes (spine density reduction and abnormally elongated spines) induced by the constitutively membrane bound S4N-MARCKS. Similarly, the substantial spine loss and shrinkage induced by MARCKS shRNA should be phenocopied by increasing endogenous PI(4,5)P2. To manipulate endogenous PI(4,5)P2 levels, we transiently overexpressed enzymes involved in PI(4,5)P2 synthesis or removal, respectively: the eGFP-tagged brain specific type Iγ isoform of phosphatidylinositol 4-phosphate 5-kinase (PIP5K; Wenk et al., 2001) and the HA-tagged inositol 5-phosphate phosphatase domain of synaptojanin 1 (5ptase; Krauss et al., 2003).

Consistent with the hypothesis, we observed the expected decrease in spine density and significant shrinkage of remaining dendritic spines in PIP5K expressing neurons, similar to that seen with shRNA-induced MARCKS silencing (Figure 2A). Furthermore, PIP5K overexpression prevented the elongation of dendritic spines induced by coexpressed membrane bound S4N-MARCKS (Figure 2B). Also, as predicted, overexpression of 5ptase caused significant lengthening of dendritic spines, similar to that induced by S4N-MARCKS (Figure 2C), and 5ptase prevented the spine shrinkage induced by MARCKS knockdown (Figure 2D). These observations demonstrate opposing morphological actions of MARCKS versus PI(4,5)P2. Note, however, that, just as previously reported for MARCKS (Calabrese and Halpain, 2005), imbalance in PI(4,5)P2 in either direction reduced overall spine density (Figure 2E; Supplemental Table S1). Interestingly, in these overexpression-based experiments, manipulations of the PI(4,5)P2 pathway appeared to predominate over that of MARCKS for the spine phenotypes, because enhancing PI(4,5)P2 availability completely prevented the usual loss and shrinkage of spines induced by MARCKS depletion, and decreasing PI(4,5)P2 availability overcame the powerful effect of excess membrane MARCKS on spine elongation (Figure 2F, Supplemental Table S1). All manipulations reduced spine head width relative to controls (Figure 2G, Supplemental Table S1).

FIGURE 2:

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Increased PI(4,5)P2 levels (PIP5K or MARCKS KD) lead to spine shrinkage, while decreased PI(4,5)P2 levels lead to spine elongation. (A) Representative dendritic regions coexpressing mcherry with either eGFP, MARCKS shRNA or PIP5K. (B) Representative dendritic regions of neurons expressing S4N-MARCKS with or without PIP5K. (C) Representative dendritic regions coexpressing mcherry with either eGFP, S4N-MARCKS or 5ptase. (D) Representative dendritic regions expressing MARCKS shRNA with or without 5ptase. All image widths = 32 µm. (E–G) Dendritic spine density, length and width for all conditions (A–D) are expressed as mean ± SEM. See Supplemental Table S1 for complete statistical information. For selected comparisons referenced in the main text: **** p < 0.0001, ***p = 0.006, ns = not significant.

PI(4,5)P2 effects on spine morphology are not mediated by PIP3 or IP3

PIP5K and 5ptase selectively affect PI(4,5)P2 over other acidic phospholipids (Cremona et al., 1999; Wenk et al., 2001). However, PI(4,5)P2 is the immediate precursor of PI(3,4,5)P3, a less abundant but also important signaling phospholipid. The protein kinase AKT is a major effector of PI(3,4,5)P3 and is known to affect dendrite and spine morphology (Kumar et al., 2005). Therefore, to test whether PIP5K effects on spine morphology were mediated by PI(3,4,5)P3 and its major effector AKT, we transfected hippocampal neurons with HA-tagged myrAKT, a constitutively active form of AKT targeted to the membrane via myristoylation. Neurons expressing myrAKT failed to show the drastic spine loss and shrinkage observed in PIP5K expressing neurons (Figure 3, A–D; Supplemental Table S1). We also assume that an effect of PI(3,4,5)P3 acting via Rac GTP exchange factors (GEFs) is unlikely to explain the effects we observed of PIP5K on spine shrinkage. Recruitment of RacGEFs is expected to increase F-actin polymerization and to induce dendritic spine enlargement, which is the opposite effect from the PIP5K-induced spine shrinkage we observe. Specifically, Rac activation through the RacGEF Tiam 1 was shown to induce spine growth during development and during LTP (Um et al., 2014; Saneyoshi et al., 2019), and RacGEF activation through Rac-Asef2 or Rac-Kalirin signaling promote dendritic spine growth and development (Cahill et al., 2009; Evans et al., 2015). Therefore, we conclude that PI(3,4,5)P3 is unlikely to mediate the PIP5K-induced changes in spine morphology, although we cannot rule out other potential actions of PI(3,4,5)P3.

FIGURE 3:

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Neither myrAKT nor IP3K reproduce the PIP5K-induced spine loss and shrinkage. (A) Representative dendritic regions of neurons expressing either eGFP or myrAKT or PIP5K together with mcherry. Image width, 32 µm. (B–D) Spine density, length and width are expressed as mean ± SEM. See Supplemental Table S1 for complete statistical information. **** p < 0.0001. (E) Representative dendritic regions of neurons expressing either eGFP, IP3K, PIP5K, or PIP5K plus IP3K. Image width = 32 µm. (F–H) Spine density, length and width are expressed as mean ± SEM; **** p < 0.0001, ns = not significant, for selected comparisons. The full set of comparisons, test selection, and statistical data are provided in Supplemental Table S1.

By similar logic we tested a degradation pathway for PI(4,5)P2. PI(4,5)P2 hydrolysis by PLC produces the second messengers diacylglycerol (DAG) and IP3 (Nahorski et al., 2003; Berridge, 2009). Enhanced production of DAG downstream of more PI(4,5)P2 synthesis would be expected to increase PKC translocation to the plasma membrane, leading to increased phosphorylation of MARCKS and thereby increasing the availability of PI(4,5)P2 (i.e., inducing a potential feed-forward loop that augments PI(4,5)P2). It is possible that enhanced PKC activity on MARCKS and other substrates mediates some of the phenotypes we observe on spines. A role for IP3 in mediating the PIP5K-induced spine shrinkage, cannot be completely excluded, although it does not appear to represent a strong influence. Depleting IP3 by overexpressing IP3 kinase, which converts IP3 to IP4 (Xia and Yang, 2005), did not by itself reproduce the PIP5K-induced spine phenotype, and it only modestly reduced the decrease in spine length caused by coexpressed PIP5K (Figure 3, E–H; Supplemental Table S1).

Together, these results indicate that two major metabolites of PI(4,5)P2 are unlikely to directly mediate the morphological effects we see on spines, suggesting that PI(4,5)P2 itself might represent the essential element mediating effects downstream of MARCKS.

PI(4,5)P2 reduces F-actin and free barbed ends (FBEs) in dendritic spines

Changes in spine shape are inextricably linked to changes in the actin cytoskeleton (Calabrese et al., 2006; Cingolani and Goda, 2008; Bosch and Hayashi, 2012; Koskinen and Hotulainen, 2014), and PI(4,5)P2 regulates the activity of several actin-binding proteins (Janmey et al., 2018; Senju and Lappalainen, 2019). We therefore tested whether the observed PI(4,5)P2-induced changes in dendritic spine morphology involve changes in the actin cytoskeleton. We aimed to capture the majority population of actin filaments in the spines by using fluorescently tagged phalloidin. F-actin is abundantly expressed in axons and astrocytes, which, by lying in close proximity to dendritic spines, impairs optimal signal-to-noise conditions for quantifying F-actin exclusive to the spine compartment. We therefore used three-dimensional image deconvolution and additional image processing methods to quantify phalloidin staining within the digitally isolated spine compartment (Figure 4, A–F; see Materials and Methods). We found that PIP5K overexpression resulted in a 34% reduction in spine F-actin concentration on average (Figure 4G; Supplemental Table S1). Remarkably, some spine heads appeared to completely or almost completely lack a detectable phalloidin signal (Figure 4G; Supplemental Figure S3, white arrows), an observation that is extremely rare in control neurons.

FIGURE 4:

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Increased PI(4,5)P2 levels reduce F-actin in dendritic spines. (A) Original image of a representative dendritic region of a neuron expressing eGFP and stained for MAP2 and F-actin. Cyan arrows: selected examples of dendritic spines. (B) Projected three-dimensional surface renderings of eGFP (green) and MAP2 (blue) fluorescence distribution. (C) Projected three-dimensional surface renderings of isolated dendritic spines obtained by subtracting MAP2 from the eGFP signal (see B). (D) Projected three-dimensional eGFP surface renderings (green) overlayed on the original F-actin image. (E) Isolated dendritic spines (white) shown in (C), overlayed on the original total F-actin signal (red). (F) F-actin only in spines, resulting from subtracting image shown in (E). See Materials and Methods for details on image processing used to measure F-actin and volume of dendritic spines. Image width = 30 µm. (G) Volumetric measurements of F-actin content per dendritic spine were calculated as the ratio of F-actin integrated intensity over spine volume. Data are expressed as mean ± SEM; *** p < 0.001. Test selection, and statistical data are provided in Supplemental Table S1.

PIP5K overexpression also induced an 82.8% reduction in the fraction of spines with detectable levels of F-actin free barbed ends (FBEs; Figure 5, A and B; Supplemental Table S1), and also reduced the average FBE content per spine (Figure 5, C and D; Supplemental Table S1). This suggests that excess PI(4,5)P2 induces an extensive decrease in initiation sites for F-actin polymerization, which could be due to increased barbed end capping or decreased barbed end production. Cofilin, an abundant actin binding protein that is reportedly inhibited when bound to PI(4,5)P2 (Yonezawa et al., 1990; van Rheenen et al., 2007; Wills and Hammond, 2022), plays a critical role in the maintenance of dendritic spine volume during resting levels of synaptic activity via its actin filament severing function (Calabrese et al., 2014). In control neurons, ongoing cofilin activity thus maintains a steady supply of FBEs and thereby supports the high rate of intrinsic spine F-actin turnover (Star et al., 2002; Honkura et al., 2008; Tatavarty et al., 2009; Koskinen et al., 2012; Colin et al., 2023).

FIGURE 5:

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Chronophin overexpression rescues spine FBE reduction induced by PIP5K. (A) Left: Control dendritic region stained with Alexa 488-phalloidin (green) to detect F-actin in dendritic spines (white arrows). Right: two examples of dendritic regions expressing eGFP-PIP5K (green). All conditions are labeled for FBEs (red) using rhodamine-G-actin. Image width, 12 µm. (B) Quantification of the fraction of protrusions with detectable FBEs. Data are expressed as mean ± SEM. See Supplemental Table S1 for complete statistical information. *** p < 0.001. (C) Representative dendritic regions of neurons expressing membrane targeted pDisplay (control), CIN + pDisplay (shown here only CIN), PIP5K, PIP5K, + CIN (for clarity only PIP5K [green] is displayed, while CIN [cyan] is included in the merged image). FBEs were labeled using Alexa 568-G-actin. Image width = 18 µm. (D) Quantification of FBE content was performed only in spines displaying FBE labeling (see Materials and Methods). Data are expressed as mean ± SEM; **** p < 0.0001, ns = not significant. The full set of comparisons, test selection, and statistical data are provided in Supplemental Table S1.

To examine a role for cofilin in the actions of PIP5K on FBEs, we increased the activity of endogenous cofilin by overexpressing the cofilin regulatory phosphatase chronophin (CIN; [Gohla et al., 2005; Huang et al., 2006; Calabrese et al., 2014; Delorme-Walker et al., 2015]). CIN dephosphorylates cofilin at its critical Ser-3 regulatory site, thereby promoting its F-actin severing activity (Gohla et al., 2005; Mizuno, 2013), and, unlike the other known cofilin phosphatase, slingshot, does not inactivate the cofilin-selective kinase LIM kinase (Huang et al., 2006). As expected, CIN expression alone induced a modest increase in spine FBEs. When coexpressed, CIN also completely prevented the dramatic loss of spine FBEs induced by PIP5K (Figure 5D; Supplemental Table S1). Together, these observations are consistent with the hypothesis that cofilin activity is inhibited by PI(4,5)P2 in the spine membrane, thereby reducing overall F-actin concentration and causing spine shrinkage and loss.

When PI(4,5)P2 levels were decreased by expressing 5ptase, we did not detect a significant change in the spine F-actin content over the entire dendrite. However, the aberrantly elongated spines induced by 5ptase often contained multiple F-actin clusters distributed along their length, instead of the single F-actin cluster at the tip that is typical of even the longest spines present in control conditions (Figure 6). This multiple actin cluster phenotype was identical to that induced by S4N-MARCKS (Calabrese and Halpain, 2005). Reductions in PI(4,5)P2 may perturb a regulatory mechanism that dictates where F-actin is clustered within dendritic protrusions.

FIGURE 6:

FIGURE 6:

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Aberrantly long protrusions induced by 5ptase contain multiple distinct clusters of F-actin along their length. (A) Dendritic regions coexpressing mRFP-actin with either eGFP (top row) or 5ptase (bottom row). Image width = 32 µm. Cyan arrows point to the actin clusters part way or at the tip of a 5ptase elongated spine. (B) Quantification of the fraction of protrusions with actin clusters only at the tip or part way. Data are expressed as mean ± SEM. Unpaired t test between eGFP and 5ptase; *** p = 0.0007. Protrusion percentage was calculated per experiment (n = 3).

Cofilin inhibition regulates the effects of PI(4,5)P2 on dendritic spine morphology

To further test the hypothesis that cofilin mediates the opposing effects of MARCKS and PI(4,5)P2 on spine morphology, we expressed PIP5K together with constructs inducing higher activity of cofilin. Consistent with the hypothesis, we found that the usual spine loss and shrinkage induced by PIP5K were reduced in the presence of CIN (Figure 7, A–D; Supplemental Table S1). The same outcome was observed when we expressed CIN together with MARCKS shRNA (Figure 7, E–H; Supplemental Table S1). Furthermore, we reversed these conditions by expressing 5ptase together with either of two conditions expected to decrease endogenous cofilin activity. First, we overexpressed LIMK to inactivate the severing activity of endogenous cofilin via phosphorylation at Ser-3. Second, we used an shRNA to silence endogenous CIN expression, thereby reducing Ser-3 dephosphorylation of cofilin. We found that both these methods of suppressing cofilin activity were able to reverse the effects of decreasing endogenous PI(4,5)P2, allowing a return of normal spine length and a reversal of spine head shrinkage (Figure 7, I–L; Supplemental Table S1). Finally, we confirmed that these “rescues” of spine morphology induced by PI(4,5)P2 depletion were also observed when PI(4,5)P2 availability was impaired via S4N-MARCKS (Figure 7, M–P; Supplemental Table S1).

FIGURE 7:

FIGURE 7:

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Increased cofilin activity rescues spine shrinkage induced by excess PI(4,5)P2, while decreased cofilin activity prevents spine elongation induced by decreased PI(4,5)P2. The availability of PI(4,5)P2 was increased via expression of either PIP5K or MARCKS shRNA; PI(4,5)P2 availability was decreased via expression of 5ptase or S4N-MARCKS; cofilin activity was increased via expression of CIN; cofilin activity was decreased via LIMK or CIN shRNA. (A) Representative dendritic regions of neurons expressing either eGFP, PIP5K, or PIP5K + CIN, together with a cell filler. Note: In these images (A, E, I, and M) only the cell filler (eGFP or mcherry) is shown. It was used for quantitative analysis of dendritic spine size. All image width = 27 µm. (B–D) Data are expressed as mean ± SEM. See Supplemental Table S1 for complete statistical information. For selected comparisons referenced in the main text: **** p < 0.0001, ***p = 0.0009, **p = 0.0028, *p = 0.0249, ns = not significant. (E) Representative dendritic regions of neurons expressing either eGFP, MARCKS shRNA, or MARCKS shRNA + CIN, together with a cell filler (See Note in A). (F–H) Data are expressed as mean ± SEM. See Supplemental Table S1 for complete statistical information. For selected comparisons referenced in the main text: **** p < 0.0001, **p = 0.001, ns = not significant. (I) Representative dendritic regions of neurons expressing either eGFP, 5ptase or 5ptase plus constructs that increase cofilin phosphorylation: LIMK, and chronophin (CIN) shRNA. (See Note in A). (J–L) Data are expressed as mean ± SEM. See Supplemental Table S1 for complete statistical information. For selected comparisons referenced in the main text: **** p < 0.0001, ***p = 0.0076, ns = not significant. (M) Representative dendritic regions of neurons expressing either S4N or S4N + LIMK. (N–P) Data are expressed as mean ± SEM. See Supplemental Table S1 for complete statistical information. For selected comparisons referenced in the main text: **** p < 0.0001, ns = not significant. Note: For display convenience, the data for the eGFP and LIMK groups are duplicated in figures J–L and N–P; however, these data were collected together within the same sets of experiments, and compared for statistical purposes using a single one-way ANOVA.

Together, these data are consistent with the idea that the effects of PI(4,5)P2 on spine morphology are mediated, at least in part, by its regulation of cofilin. They are consistent with the idea that at steady state MARCKS partially suppresses PI(4,5)P2 availability, thereby allowing a population of cofilin to remain constitutively active to maintain normal spine shape and volume. Note, however, that the cofilin manipulations only partially restored normal spine densities in any condition, suggesting that other factors, in addition to cofilin, contribute to this phenotype.

DISCUSSION

In this study we have demonstrated important functional interactions between the phospholipid PI(4,5)P2 and the actin regulatory protein cofilin as a basis for the previously reported effects of MARCKS on the morphology of dendritic spines (Calabrese and Halpain, 2005). This trio of relations among MARCKS, PI(4,5)P2, and cofilin supports a model in which all three play vital roles in the maintenance of mature dendritic spines, (i.e., in spines that have already exhibited their adult morphology and participate in synaptic plasticity). Each of these players has been independently implicated in the mechanisms of synaptic plasticity (Calabrese and Halpain, 2005; McNamara et al., 2005; Timofeeva et al., 2010; Trovo et al., 2013; Brudvig and Weimer, 2015; Senju and Lappalainen, 2019; Mandal, 2020; Bura et al., 2023), but their functional interactions in this context have yet to be explored. Our new observations reinforce the view that, at the molecular level, membrane bound MARCKS, mediates its physiological effects on spine morphology mainly, if not exclusively, by decreasing the availability of PI(4,5)P2 to its downstream effectors (Glaser et al., 1996; Sundaram et al., 2004). Furthermore, we provide compelling evidence that the actin binding protein cofilin is an important mediator of the effects of PI(4,5)P2 and MARCKS on dendritic spine morphology. These new findings are summarized in a diagram in Figure 8.

FIGURE 8:

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Summary diagram for the role of MARCKS and PI(4,5)P2 interactions in controlling dendritic spine morphology through cofilin activity. Our experiments support a model in which changes in MARCKS levels at the membrane affect the amount of free PI(4,5)P2 that is available to suppress cofilin severing activity. In turn this affects spine shape and size via F-actin. Regulation of membrane trafficking was not explored here, but is also likely to contribute to spine modifications.

MARCKS and PI(4,5)P2 are both important targets for regulation by intracellular signaling. MARCKS is driven off the plasma membrane either via multisite phosphorylation by PKC within its polybasic effector domain (Wu et al., 1982), or by the binding of Ca2+/calmodulin to the effector domain (Arbuzova et al., 2002). Either type of calcium-dependent signal interrupts the electrostatic interaction between MARCKS and PI(4,5)P2, thereby freeing PI(4,5)P2 from sequestration by MARCKS (Glaser et al., 1996; Sundaram et al., 2004). On the other hand, PI(4,5)P2 is down-regulated by PLC in response to various stimuli (Bill and Vines, 2020). PLC hydroylzes PI(4,5)P2 into the second messengers DAG and inositol triphosphate (IP3), thereby depleting PI(4,5)P2 concentrations while also releasing intracellular stores of Ca2+ and activating PKC. These interactions are coupled into feedback and feedforward regulatory loops, not least because the binding of MARCKS to PI(4,5)P2 inhibits the enzymatic activity (but possibly not the binding) of PLC (Gambhir et al., 2004). In other words, any activity of PLC, not inhibited directly by the binding of MARCKS to PI(4,5)P2, would generate a feedback effect to limit the inhibitory action of MARCKS on PLC whenever PLC is activated. We assume that under the basal, nonstimulated conditions we used in our experiments PLC activity remains relatively low, as described in other model systems (Bill and Vines, 2020). The precise role of regulatory pathways on the MARCKS and PI(4,5)P2 effects we observe on spines remain to be further investigated.

By using a selective PI(4,5)P2 binding probe (the PH domain of PLCδ1) as a reporter of free PI(4,5)P2 on the membrane, and quantifying a MAI, we provide evidence for a reciprocal relationship between plasma membrane-bound MARCKS and free plasma membrane PI(4,5)P2. The simplest explanation for our observation that the level of “membrane-competent” MARCKS anticorrelates with the binding of the mRFP-PHPLCδ1 probe is that MARCKS competes successfully with the PH domain of PLC for binding to PI(4,5)P2 in intact neuronal cells. However, as the experiments by Gambhir et al. (2004) suggest that the MARCKS effector domain does not inhibit the binding of eGFP-PH-PLCδ1 to large unilamellar vesicles containing PI(4,5)P2, we cannot completely rule out that full length eGFP-S4N-MARCKS might bind mRFP-PHPLCδ1-bound PI(4,5)P2 in intact neurons.

MARCKS and PI(4,5)P2 are both present in spines, and there is no evidence that the electrostatic interaction between MARCKS and PI(4,5)P2 would differ between the shaft and spine compartments. Moreover, Hofbrucker-MacKenzie et al. (2023) provided evidence that endogenous PI(4,5)P2 is regulated similarly in the dendritic spine and shaft compartments in response to calcium changes. Therefore, despite technical limitations that prevented us from performing MAI measurements within the small compartment of spines, we postulate that the same reciprocal relationship prevails in spines as in dendrite shafts. Future superresolution and/or quantitative EM studies may be useful in probing such dynamics within the spine compartment specifically.

We interpret our findings on the downstream effects of MARCKS on spine morphology to be mainly due to its direct and potentially super-stoichiometric inhibition of PI(4,5)P2. Each effector domain of MARCKS is capable of binding up to three molecules of PI(4,5)P2 (Glaser et al., 1996), suggesting that in cells MARCKS can exert a powerful effect on the PI(4,5)P2 signaling machinery.

PI(4,5)P2 is of particular importance in regulating events at the cell periphery, especially vesicle insertion/retrieval and the arrangement and activities of the actin cytoskeleton, which itself is a hub for organizing cellular functions (Senju and Lappalainen, 2019). PI(4,5)P2 binds and regulates multiple actin binding proteins, as well as some small GTPases that, in turn, regulate actin. Typically, PI(4,5)P2 activates proteins that promote F-actin assembly, like N-WASP and certain diaphanous-family formins, and it inhibits proteins that promote F-actin disassembly, (i.e., the cofilin-family proteins ADF/cofilin/twinfilin). As a consequence of these combined forces, an increase in free PI(4,5)P2 will usually induce a net gain in actin polymerization beneath the membrane (van Rheenen et al., 2007; Frantz et al., 2008; Van Troys et al., 2008; Senju and Lappalainen, 2019). The same holds true for the PI(4,5)P2 metabolite PI(3,4,5)P3 acting via RacGEFs (Welch et al., 2003; Campa et al., 2015). Intriguingly, however, because we observe that a PIP5K-induced increase in free PI(4,5)P2 causes spine shrinkage rather than spine growth, the mechanisms regulating F-actin in spines may differ in key respects from how F-actin is regulated in other contexts. This potential distinction warrants further study.

The role of cofilin in the balance between F-actin assembly and disassembly is complex. Cofilin has robust F-actin severing activity in its dephosphorylated state, and also facilitates the removal of actin monomer from the recently severed pointed end (Mizuno, 2013). However, dephosphorylated cofilin should not be thought of merely as a “depolymerizing factor.” In many cellular conditions, these activities of cofilin are exploited to “dynamize” the actin cytoskeleton and thereby ensure a steady supply of FBEs and recycled monomers for reuse in actin polymerization near the plasma membrane (Bravo-Cordero et al., 2013; Bamburg et al., 2021). Indeed, we postulate that shifting the balance toward a dephosphorylated cofilin population provides enough “excess” cofilin activity throughout the cytoplasm to overcome the ability of free PI(4,5)P2 to capture it at the membrane, thereby evoking a robust rescue of the PIP5K phenotype by CIN.

In neurons, acute depletion of endogenous cofilin results in a substantial shrinkage and loss of spines (Calabrese et al., 2014). These prior results, and those reported here, are consistent with a model in which, under control conditions, cofilin is moderately active to maintain dynamic spine F-actin turnover. A proper balance in PI(4,5)P2 availability contributes to the moderate activity levels of endogenous cofilin, most of which in dendritic spines is localized adjacent to the plasma membrane (Racz and Weinberg, 2006). The regulation of cofilin phosphorylation by protein kinases and phosphatases also play critical roles. Our results suggest that cofilin inactivation by PI(4,5)P2 serves to reduce the normal levels of FBEs and total F-actin that ordinarily maintain spine volume during basal conditions.

Taken all together, our results from the experimental manipulations of MARCKS, free PI(4,5)P2, and cofilin suggest that the relative activity of these three elements has a substantial impact on the maintenance of dendritic spine morphology. Imbalance in any of these three factors induces detectable and often dramatic effects on spine density and shape. Membrane bound MARCKS decreases the availability of free PI(4,5)P2, which, in turn, allows more F-actin severing activity by cofilin. Under control conditions, cofilin maintains a steady supply of FBEs that support dynamic turnover of spine F-actin to maintain normal spine shape and volume (Calabrese et al., 2014). Our current and prior data suggest that a careful balance between free PI(4,5)P2 versus membrane-bound MARCKS helps sustain cofilin activity at optimal levels for normal maintenance of actin properties and spine morphology, which, in turn, are likely to regulate the concentration of glutamate receptors and the activity of synapses (Harris and Stevens, 1989; Arellano et al., 2007; Kasai et al., 2010; Borczyk et al., 2019).

Bidirectional modulation of cofilin activity via a PI(4,5)P2-dependent pathway might also play a role in structural plasticity. A role for PI(4,5)P2 in the NMDA-dependent induction of LTP and LTD has been investigated (Horne and Dell’Acqua, 2007; Gong and De Camilli, 2008; Suh and Hille, 2008; Unoki et al., 2012; Trovo et al., 2013; Hofbrucker-MacKenzie et al., 2023). Here, we found that incubation of cultures with NMDA stimulated a transient increase in free PI(4,5)P2, followed by a prominent depletion. Our data align with a recent elegant study using immunogold labeling of PI(4,5)P2 and quantitative freeze-fracture electron microscopy, which similarly reported that a brief incubation of cultured neurons with NMDA (an LTD-like condition), was associated with a transient increase in spine PI(4,5)P2 (Hofbrucker-MacKenzie et al., 2023). The authors also provided convincing evidence that NMDA increased PI(4,5)P2 levels via PIP5K-dependent synthesis, although other potential mechanisms were not excluded. It seems likely, for example, that NMDA would stimulate a PKC-dependent decrease in membrane bound MARCKS (Etoh et al., 1991; Hartwig et al., 1992; Verghese et al., 1994; Calabrese and Halpain, 2005), which might also contribute to a burst of freely available PI(4,5)P2. These findings are consistent with a model in which NMDA receptor activation drives spine shrinkage by increasing free PI(4,5)P2, decreasing steady-state cofilin activity near the spine plasma membrane, and decreasing the FBEs required for maintaining spine volume.

Additional actin-based and membrane trafficking mechanisms are likely to contribute to spine shrinkage as well. It is important to recognize that, although a direct inhibition of cofilin activity by PI(4,5)P2 would supply the simplest explanation for our observations, it is possible that indirect actions of PI(4,5)P2 may be at play. Indeed, the binding affinity between cofilin and PI(4,5)P2-containing membranes is weak compared with that between PI(4,5)P2 and other actin binding proteins (Senju et al., 2017), and further research is needed to more fully understand the downstream physiological actions of a PI(4,5)P2 increase or a MARCKS decrease.

Despite a central role for cofilin implied by the present results, it seems unlikely that cofilin would be the sole mediator of PI(4,5)P2’s influence on spine morphology. While cofilin can profoundly influence F-actin turnover dynamics in spines (Hotulainen et al., 2009; Calabrese et al., 2014; Spence and Soderling, 2015), other factors might be needed to regulate F-actin polymerization and/or the trafficking of membrane within the spine. Undoubtedly, PI(4,5)P2 regulation of effectors like profilin, actin capping proteins, the ezrin/radixin/moesin (ERM)-family proteins, etc., must also be considered.

In contrast to spine shrinkage, where many potential mechanisms are known, the aberrantly long spine protrusions induced by either MARCKS-S4N or 5ptase (either of which should restrict plasma membrane PI(4,5)P2 availability) are more difficult to fully understand at present. Our observations show that inhibiting endogenous cofilin activity can strongly reduce the abnormal elongation of spines caused by increased 5ptase or increased MARCKS, implying that excess cofilin activity is a significant consequence of decreased PI(4,5)P2. This relationship might be direct or indirect, although a direct inhibition of cofilin by its binding to PI(4,5)P2 seems plausible. Other PI(4,5)P2-regulated proteins likely contribute to the pronounced elongation of spines, as well as to the decrease in overall protrusion density, which our prior work on MARCKS suggested may stem from spines fusing with one another (Calabrese and Halpain, 2005). A decrease in the focal control of actin polymerization might also play a role, as suggested by the effects of MARCKS-S4N in destabilizing the focal clustering of F-actin within the aberrantly elongated spines (Calabrese and Halpain, 2005). Additionally, decreased anchoring of membrane PI(4,5)P2 to ERM proteins might create an abnormally fluid membrane that can be more readily stretched or fused (Bretscher et al., 2000; Hao et al., 2009). Finally, an imbalance of endocytosis and exocytosis, which are both known to be regulated by PI(4,5)P2 (Koch and Holt, 2012; Martin, 2012; Brown, 2015), is also likely to contribute to a profound perturbation in dendritic spine morphology (Racz et al., 2004; Park et al., 2006).

The present report focuses on the regulation of spine morphology in neuronal dendrites that possess existing synaptic connections and nominally mature neuronal morphology. However, the cellular and molecular mechanisms we describe here are presumably relevant to other aspects of neuronal function and development, as well as to physiological processes in other cells and tissues. Cofilin, MARCKS, and PI(4,5)P2 metabolism have each been implicated in genetic perturbations causing a range of neurological disorders, and are broadly implicated in neurodegeneration, aging, cancer, inflammation, airway diseases, and other conditions (Pinner et al., 2014; Sheats et al., 2019; Alsegiani and Shah, 2020; Theis et al., 2020; Chiu et al., 2022; Lv et al., 2022). It may be useful to consider the molecular mechanisms we propose here as potential contributors to various disease phenotypes.

MATERIALS AND METHODS

Request a protocol through Bio-protocol.

Plasmids

mRFP-actin and MARCKS shRNA were previously described and validated in Calabrese and Halpain (2005); all constructs were confirmed by DNA sequencing. The following reagents were generous gifts from colleagues: MyrAKT (K.C. Arden; UC San Diego; validated by Biggs et al. [1999]); mRFP-PHPLCδ1 (T. Balla, NIH; validated by Varnai and Balla (1998); PSD95-GFP (lA. El Husseini, University of British Columbia); GFP-PIP5K Iγ (P. De Camilli, Yale University; enzymatic activity validated by Wenk et al. (2001); HA-tagged inositol 5-phosphate phosphatase domain of synaptojanin 1 (5ptase; V. Haucke, University of Göttingen; enzymatic activity validated by Wenk et al. [2001] and Krauss et al. [2003]); IP3K (M. Schell, Uniformed Services University, Bethesda; enzymatic activity validated by Yu et al. [2005]); GFP-actin (A. Matus, Friedrich Meinscher Institute, Basal); pDisplay (A. Ghosh, UC San Diego); full length wild type rat LIMK was a kind gift by G. Bokoch (TSRI), validated by (Gohla and Bokoch, 2002); pEGFP-N1 was obtained from Clontech.

mcherry was received as pRSETBmcherry (gift of R.Tsien).To express it in mammalian cells we cut the mcherry containing fragment using EcoRI, followed by Klenow and Bam H1 and we inserted it into peGFP-N1 backbone after removing eGFP with NotI, followed by Klenow and BamHI. S4N-MARCKS-CFP was generated by digesting eGFP-tagged S4N-MARCKS (gift of P. Blackshear) with NheI and AgeI; the resulting S4N-MARCKS containing fragment was filled in with Klenow to produce blunt ends and ligated into pCFP-N1, cut with SmaI. Please refer to Swierczynski and Blackshear (1995) for the validation of the original S4N-MARCKS construct, there called tetra-Asn or TN. Rat HA- chronophin (CIN), was generated in our laboratory by J. Lauterbach and previously described and validated (Calabrese et al., 2014). CIN-shRNA was generated by J. Lauterbach (Halpain laboratory). The following oligonucleotides were annealed and inserted into the HindIII/BglII sites of the pSuper-eGFP vector used in Calabrese and Halpain (2005): CIN-shRNA, 5′-GAT CCC CGC ATG CTG ATG GTG GGA GAT TCA AGA GAT CTC CCA TCA GCA TGC TTT TTG GAA A-3′, and 5′-AGC TTT TCC AAA AAG CAT GCT GAT GGT GGG AGA TCT CTT GAA TCT CCC ACC ATC AGC ATG CGG G -3′ (corresponding to nucleotides 368–386); The shRNA construct was expressed under control of the polymerase- III H1-RNA gene promoter (Brummelkamp et al., 2002).

The efficacy of the two newly generated CIN shRNA constructs was assessed by overexpressing exogenous CIN in P19 cells either on its own or together with each of the two CIN shRNA constructs (Supplemental Figure S4A). CIN shRNA #2 performed significantly better than CIN shRNA #1 in preventing CIN overexpression (Supplemental Figure S4B). Indeed, CIN shRNA #2 was also confirmed to knock down endogenous CIN within 7 d in vitro (DIV) hippocampal neurons (Supplemental Figure S5).

Cell culture and transfection

Hippocampal cultures were prepared according to Calabrese and Halpain (2005) at a density of 300 cells/mm2 and maintained in neurobasal medium (GIBCO), supplemented with B27 (Invitrogen) and 0.5 mM L-glutamine (Sigma). Neurons were transfected at 20 d in vitro (DIV) using calcium phosphate precipitation. Solutions and the range of cDNA concentrations were chosen according to Kohrmann et al. (1999). Cells were incubated with the transfection mixture for 3 h in a 5% CO2 incubator at 37°C, washed twice with prewarmed HEPES buffered saline (HBS) solution (in mM: 135 NaCl, 4 KCl, 1 Na2HPO4, 2 CaCl2, 1 MgCl2, 10 glucose, and 20 HEPES, [pH, 7.35]) and replaced in the medium in which they had been growing before transfection. Neurons were fixed or used for live cell-imaging experiments 24–48 h posttransfection, except for the CIN shRNA experiments when neurons were fixed 4 d posttransfection.

COS-7 cells were maintained in Dulbecco’s modified Eagle’s high glucose medium (Life Technologies-BRL) supplemented with 10% fetal bovine serum (Life Technologies). Cell passaging was performed using trypsin-EDTA for 1–2 min at 37°C. Cells were transfected after 7–8 h from trypsinization using Lipofectamine 2000 (Invitrogen). One to two days after cells were observed by fluorescent microscopy. P19 cells were transfected using FuGENE 6 (Roche Molecular Biochemicals).

NMDA-induced spine loss was induced according to (Lee et al., 1998; Calabrese et al., 2014). Briefly hippocampal neurons were incubated with 40 µM NMDA (Sigma) for 4 min prior fixation.

Time-Lapse Recordings

Neurons were cultured and transfected on Lab-Tek II chambered coverglass (155409; Nalge Nunc International). Live images were acquired every 10 min over 1 h period with exposures of 0.1– 0.5 s using an Olympus IX-70 microscope equipped with a customized CO2-gassed, temperature-controlled chamber (5% CO2, 35°C). Images were collected at 60X using a Cool SNAP HQ2 camera (Nikon).

Free barbed end assay

For the labeling of actin nucleation sites (barbed ends) rhodamine labeled nonmuscle G-actin protein (Cytoskeleton) was diluted in 1 mM HEPES pH 7.5, 0.2 mM MgCl2, 0.2 mM adenosine triphosphate (ATP), sonicated briefly and centrifuged at 4°C at a speed of 50 K rpm for 10 min using a TLA-100.3 rotor (Beckman). Then cells were permeabilized for 1.5 min at 37°C with the supernatant containing rhodamine G-actin plus 1% bovine serum albumin (BSA), 0.25 mg/ml saponin, 20 mM HEPES, 138 mM KCl, 4 mM MgCl2, 9 mM EGTA, and 1 mM ATP before being fixed with 3.7% formaldehyde. A final concentration of 0.85 µM G-actin was used per each 12-mm coverslip. This assay was previously validated in nonneuronal cells (Shestakova et al., 2001) and cultured hippocampal neurons (Calabrese et al., 2014).

Image analysis and quantification

Prior to image collection and analysis, sample identity was encoded so that the manipulation category was “blind” to the observer. Fluorescence images were collected with either an Olympus Fluoview 500 confocal microscope by sequential illumination using the 488 nm line of an argon laser, and the HeNe Green 543 nm, HeCd 442 nm lasers or a CSUX1 spinning disk confocal (Yokogawa) mounted on an Olympus IX70 with a 491 nm, 561 nm, and 647 nm solid laser lines (Solamere). Sequential acquisition and the use of a bandpass filter (BA 505/525) eliminated bleed-through between the eGFP and the CFP channel. A stack of images was acquired in the z dimension at optical slice thickness of 0.2–0.4 µm to capture entire neurons, using a 60 × 1.4 NA Plan APO oil immersion objective.

The coefficient of variation was calculated as the ratio between the SD over the mean of pixel intensity of the dendritic region.

The MAI was calculated using ImageJ. Multiple linescans were traced across the dendritic shaft of neurons expressing mRFP-PHPLCδ1. A resulting intensity profile characterized by two peaks and a trough indicated that mRFP-PHPLCδ1 was distributed mainly on the plasma membrane, while the lack of a trough suggested a cytosolic distribution. The ratio between the average peak intensities and the intensity value half-way between the two peaks represented the MAI.

The volume of F-actin per dendritic spine was calculated using IMARIS software. The three-dimensional rendering (iso-surface) of MAP2 staining was used to create a mask of the dendritic shaft which was digitally subtracted from the rest of the dendrite to leave only the dendritic spine compartment. Then the integrated intensity of the overlaid F-actin signal was determined within the three-dimensional spine volume (Figure 4A; Supplemental FigureS3).

The relative concentration of FBEs per dendritic spine were quantified by dividing the integrated intensity of the G-actin signal by the area of the spine compartment (Calabrese et al., 2014).

For the detailed protocol used to quantify dendritic spine length and width, please refer to Calabrese and Halpain (2005).

The quantification of the colocalization of the PSD95 and PH clusters in neurons transfected with these two constructs was achieved using MetaMorph imaging software (Universal Imaging Corporation, WestChester, PA). Confocal images of dendrites were thresholded to highlight clusters according to user-defined settings, using the same image display settings for all treatment groups within an experiment. Cluster area and colocalization coefficients of one probe over the other and vice versa were calculated automatically by the MetaMorph software.

Immunocytochemistry

Neurons were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) plus 120 mM sucrose for 20 min at 37°C. Neurons were incubated in 20 mM glycine for 5 min, rinsed and permeabilized with 0.2% Triton X-100 for 5 min at room temperature, and then blocked for 30 min with 2% BSA. Mouse monoclonal anti-CIN antibody at 1:200 (clone SVP-38, Sigma), anti-LIMK 1:1000 (obtained from G. Bokoch) and rat anti-HA 1:500 (Roche) and rabbit polyclonal MAP2 antibody 1:3000 (Ozer and Halpain, 2000) were incubated for 1 h at room temperature, and, following rinsing, were incubated with AlexaFluor-conjugated secondary antibodies (Molecular Probes) for 45 min at 37°C. To label F-actin, AlexaFluor 488-, 568-, or 647-phalloidin at 1:1000 (Molecular Probes) was incubated for 2 h at room temperature in the presence of 2% BSA.

Data analysis

Statistical calculations (Student’s t test, one-way and two-way ANOVA) were performed in Graphpad Prism. Significance was set at p < 0.05. Whenever the data did not fit a Gaussian distribution, a nonparametric test was performed.

Supplementary Material

mbc-35-ar23-s001.pdf (1.1MB, pdf)

Acknowledgments

We thank Brian Jenkins and Catherin Nguyen for technical support, W.B. Kiosses for training on the IMARIS software, and the many colleagues who generously supplied reagents. We thank J. Lauterbach for creating the HA-CIN and CIN shRNA used in this study, and Halpain lab members for inspiring discussions.

This work was supported by National Institutes of Health NS37311 and MH087823 (S.H.); Air Force Office of Scientific Research, grant FA9550-18-1-0051 (S.H.); American Health Assistance Foundation (B.C.).

Abbreviations used:

ATP

adenosine triphosphate

CIN

chronophin

F-actin

filamentous actin

FBEs

free barbed ends

GEFs

GTP exchange factors

HBS

HEPES buffered saline

IP3K

inositol 1,4,5-triphosphate 3-kinase

LIMK

LIM kinase

LTD

long term depression

LTP

long term potentiation

MAI

membrane association index

MARCKS

myristoylated alanine-rich C kinase

myrAKT

myristoylated AKT/protein kinase B

NMDA

N-methyl- d-aspartate

PH PLCδ1

pleckstrin homology domain of phospholipase C δ1

PI(3,4,5)P 3

phosphatidylinositol-3,4,5-triphosphate

PI(4,5)P 2

phosphatidylinositol-4,5-biphosphate

PIP5K

phosphatidylinositol-4-phosphate 5-kinase

PKC

protein kinase C

5ptase

inositol polyphosphate 5-phosphate

Footnotes

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E23-09-0370) on December 13, 2023.

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