Cargo- and adaptor-specific mechanisms regulate clathrin-mediated endocytosis

Dab2-mCherry colocalizes with LCa-EGFP and σ2-EGFP in CCPs at the cell surface.

“Dab2-mCherry colocalized with enhanced GFP (EGFP)–tagged clathrin light chain a (LCa-EGFP; Fig. 1, A–C) and EGFP-tagged σ2 subunit of AP2 (σ2-EGFP; Fig. S2 A) in CCPs at the cell surface.”

Under these conditions, ARH partially displaces AP2 from CCPs.

“Under these conditions, ARH partially displaces AP2 from CCPs”

In cells expressing CD8/LDLR, ARH-mCherry displayed nearly identical accumulation kinetics during the first 30-40 s at CCPs as compared with LCa-EGFP.

“In cells expressing CD8/LDLR, ARH-mCherry displayed nearly identical accumulation kinetics during the first 30–40 s at CCPs as compared with LCa-EGFP (Fig. 8 D, orange lines vs. green lines, respectively).”

If CD8/LDLR is first expressed for 2-7 h and then LmB is added, ARH redistributes from the nucleus to CCPs.

“However, if we first expressed CD8/LDLR for 2–7 h and then added LmB, ARH redistributed from the nucleus to CCPs (Fig. S3 Fb,c).”

The effect of CD8/LDLR overexpression on CCP dynamics was relatively small.

“the effect of CD8/LDLR overexpression on CCP dynamics was relatively small.”

In the presence of CD8/LDLR, ARH, which is lacking NPF motifs, did not induce formation of flat lattices.

“(ii) ARH, which is lacking NPF motifs, did not induce formation of flat lattices”

ARH translocated from the nucleus into the cytosol upon CD8/LDLR expression.

“ARH translocated from the nucleus into the cytosol upon CD8/LDLR expression”

These various adaptors strongly colocalize in CCPs.

“(i) the strong colocalization of these various adaptors in CCPs”

ARH expression induces strong membrane recruitment of AP2 even in the absence of CD8/LDLR overexpression.

“Interestingly, we found that ARH expression induced strong membrane recruitment of AP2 even in the absence of CD8/LDLR overexpression (Fig. 5 D, µ2 signal)”

A small fraction of nonterminal endocytic events is detected in BSC1 cells expressing Dab2.

“However, we were also able to detect a small fraction of “nonterminal” events (Fig. 3 A, arrowhead at 140 s)”

At very high levels of CD8/LDLR overexpression, Dab2 induced the formation of large, flat, and endocytically inactive clathrin lattices on the ventral cell surface.

“At very high levels of CD8/LDLR overexpression, Dab2 induced the formation of large, flat, and endocytically inactive clathrin lattices on the ventral cell surface.”

With increasing expression levels of CD8/LDLR, the efficiency of CME decreases.

“With increasing expression levels of CD8/LDLR, the efficiency of CME decreased (Fig. 6 A).”

CCPs detected by EM on the dorsal surface of BSC1 cells overexpressing TfnR alone are smaller than in control cells.

“Indeed, in our studies, CCPs detected by EM on the dorsal surface of BSC1 cells overexpressing TfnR alone were smaller than in control cells (Table I).”

The endocytic efficiency of CD8/LDLR uptake under high expression levels is increased in cells expressing an additional pool of ARH.

“However, the endocytic efficiency of CD8/LDLR uptake under high (0 ng/ml tet) expression levels was increased in cells expressing an additional pool of ARH (Fig. 6, B and C; open symbols/bars).”

Both Dab2 and ARH increased the size of CCPs relative to CCPs in which AP2 is the predominant adaptor (i.e., under conditions of TfnR overexpression).

“The fact that both Dab2 and ARH increased the size of CCPs relative to CCPs in which AP2 is the predominant adaptor (i.e., under conditions of TfnR overexpression)”

At very high levels of expression, most of the CD8/LDLRs are diffusely distributed on the plasma membrane and not captured in CCPs.

“At very high levels of expression, most of the CD8/LDLRs are diffusely distributed on the plasma membrane and not captured in CCPs.”

ARH displaces AP2 from cargo-laden CCPs.

“(iii) the displacement of AP2 by ARH from cargo-laden CCPs.”

Clathrin-coated pit size and dynamic behavior varies with LDLR expression levels in a manner dependent on the LDLR-specific adaptors Dab2 and ARH.

“Clathrin-coated pit size and dynamic behavior varies with low density lipoprotein receptor (LDLR) expression levels in a manner dependent on the LDLR-specific adaptors, Dab2 and ARH.”

Exogenously expressed mCherry-labeled Dab2 colocalizes with clathrin-coat components in control cells.

“In agreement with previous studies (Morris and Cooper, 2001; Keyel et al., 2006; Maurer and Cooper, 2006; Chetrit et al., 2009), we observed colocalization of exogenously expressed mCherry-labeled Dab2 with clathrin-coat components in control cells.”

• cites Morris and Cooper, 2001 sourceNot checked yet
• cites Keyel et al., 2006 sourceNot checked — no full text of the source
• cites Maurer and Cooper, 2006 sourceNot checked — no full text of the source

CD8/LDLR overexpression alone does not significantly alter overall CCP density.

“Consistent with this, CD8/LDLR overexpression alone does not significantly alter overall CCP density (Fig. S4 A).”

In the presence of CD8/LDLR, Dab2-p67, which is unable to bind clathrin and AP2 but contains NPF motifs, was able to induce the formation of GCCSs.

“(i) Dab2-p67, which is unable to bind clathrin and AP2 but contains NPF motifs, was able to induce the formation of GCCSs”

Increased LDLR/ARH levels led to a slight increase in the initiation, turnover rate, and number of abortive CCPs.

“increased LDLR/ARH levels led to a slight increase in the initiation, turnover rate, and number of abortive CCPs.”

At endogenous levels of cargo, there was an increase in the number of nonterminal endocytic events.

“At endogenous levels of cargo, we observed an increase in the number of nonterminal endocytic events”

Under the same experimental conditions, ARH displayed a fundamentally distinct intracellular distribution: it was predominantly nuclear with only a barely detectable recruitment to CCPs.

“Under the same experimental conditions, ARH displayed a fundamentally distinct intracellular distribution: it was predominantly nuclear with only a barely detectable recruitment to CCPs.”

Strong membrane recruitment of myristoylated Dab2 alone is sufficient to induce GCCSs even in the absence of exogenously expressed cargo.

“Even in the absence of exogenously expressed cargo, the strong membrane recruitment of this protein alone was sufficient to induce GCCSs (Fig. 2 Aa)”

Overexpression of CD8/LDLR increased the recruitment of both Dab2 and ARH to the plasma membrane.

“overexpression of CD8/LDLR increased the recruitment of both Dab2 and ARH to the plasma membrane.”

In cells overexpressing CD8/LDLRs, with or without additional ARH, the relative contribution of productive CCPs decreases to 41.3 ± 2.1% and 44.5 ± 2.2%, respectively.

“Surprisingly, in cells overexpressing CD8/LDLRs in either the absence or presence of an additional pool of ARH, the relative contribution of productive CCPs decreased to 41.3 ± 2.1% and 44.5 ± 2.2%, respectively (Fig. 7 A).”

Overexpression of LDLR slows the rate of CCP maturation.

“slows the rate of CCP maturation”

The combined presence of ARH and CD8/LDLR does not induce formation of GCCSs.

“Unlike Dab2, the combined presence of ARH and CD8/LDLR did not induce formation of GCCSs.”

Like LCa-EGFP and ARH-mCherry, AP2 rapidly accumulated in CCPs.

“Like LCa-EGFP and ARH-mCherry, AP2 rapidly accumulated in CCPs (Fig. 8 E, blue lines).”

The increased efficiency of LDLR uptake is accomplished, in part, by a prolonged growth phase and increased size of individual CCPs.

“Our live-cell and electron microscopy experiments demonstrated that this is accomplished, in part, by a prolonged growth phase and increased size of individual CCPs.”

TfnR uptake, which is also saturated upon overexpression, is not rescued by ARH.

“To establish the specificity of this effect, we showed that TfnR uptake, which was also saturated upon its overexpression, was not rescued by ARH (Fig. S1 C).”

Overexpression of LDLR increases the size of CCPs through membrane recruitment of its specific adaptors, Dab2 and ARH.

“and increases the size of CCPs through membrane recruitment of its specific adaptors, Dab2 and ARH.”

Overexpression of LDLR increases the rate of initiation and turnover of abortive CCPs.

“In contrast, we show here that overexpression of another constitutively internalized cargo, the LDLR, increases the rate of initiation and turnover of abortive CCPs”

Expression of an additional pool of ARH in the presence of CD8/LDLR reduced the amount of σ2-EGFP per CCP.

“Expression of an additional pool of ARH in the presence of CD8/LDLR reduced the amount of σ2-EGFP per CCP (Fig. 8, E and F; green lines and bars, respectively).”

The average lifetime of late abortive structures decreases from 19.1 ± 1.7 s under control conditions to 17.2 ± 1.2 s with CD8/LDLRs and further to 15.5 ± 0.8 s upon ARH expression.

“the average lifetime of late abortive structures was decreased from 19.1 ± 1.7 s under control conditions to 17.2 ± 1.2 s in the presence of CD8/LDLRs and further decreased to 15.5 ± 0.8 s upon expression of ARH (Fig. 7 B).”

The vast majority of ARH-mCherry is found in the nucleus of BSC1 cells.

“To our surprise, unlike the identically labeled Dab2, the vast majority of ARH-mCherry was found in the nucleus of BSC1 cells (Fig. 3, A and B).”

ARH is able to increase the efficiency of LDLR uptake at saturation.

“Here, we have taken care to select stable transformants expressing low levels of ARH-mCherry and show biochemically that ARH is able to increase the efficiency of LDLR uptake at saturation.”

There is a cargo and adaptor expression-dependent continuum in regulating the size of CCPs, ordered as TfnR overexpression < control cells = Dab2 alone < CD8/LDLR overexpression alone < low CD8/LDLR + Dab2 < high CD8/LDLR + ARH ≪ high CD8/LDLR + Dab2.

“Thus, we observed a cargo and adaptor expression-dependent continuum in regulating the size of CCPs, such that TfnR overexpression < control cells = Dab2 alone < CD8/LDLR overexpression alone < low CD8/LDLR + Dab2 < high CD8/LDLR + ARH ≪ high CD8/LDLR + Dab2.”

The average lifetime of productive CCPs increases significantly from 62.8 ± 3.1 s to 75.5 ± 5.0 s upon CD8/LDLR overexpression.

“the average lifetime of productive CCPs, which reflects the rate of maturation, increased significantly from 62.8 ± 3.1 s to 75.5 ± 5.0 s upon CD8/LDLR overexpression (Fig. 7 B).”

In cells expressing Dab2-mCherry, the size of CCPs increased with increasing levels of CD8/LDLR expression.

“In cells expressing Dab2-mCherry, the size of CCPs increased with increasing levels of CD8/LDLR expression.”

Nuclear exit of ARH is passive.

“Together, these results show that nuclear exit of ARH is passive and suggest the existence of a second PTB domain binding partner required for nuclear import of ARH.”

Saturated CME can be partially accounted for by a limited amount of adaptor proteins.

“Furthermore, these results demonstrate that saturated CME can be partially accounted for by a limited amount of adaptor proteins.”

The observed heterogeneity in the dynamic behavior of CCPs could reflect differences in their content of cargo and adaptor molecules.

“Together, these data strongly suggest that the observed heterogeneity in the dynamic behavior of CCPs (Merrifield et al., 2005; Rappoport et al., 2005; Yarar et al., 2005) could reflect differences in their content of cargo and adaptor molecules.”

• cites Merrifield et al., 2005 sourceNot checked — no full text of the source
• cites Rappoport et al., 2005 sourceCheck pending
• cites Yarar et al., 2005 sourceCheck pending

The relative amounts of ARH and AP2 define the size (i.e., the intensities) of diffraction-limited CCPs.

“and that the relative amounts of ARH and AP2 define the size (i.e., the intensities) of diffraction-limited CCPs.”

Constitutive CME is saturable.

“Previously, it has been reported that constitutive CME is saturable (Warren et al., 1997, 1998; Loerke et al., 2009).”

• cites Warren et al., 1997 sourceCheck pending
• cites Warren et al., 1998 sourceNot checked — no full text of the source
• cites Loerke et al., 2009 sourceCheck pending

Reduced CME efficiency under CD8/LDLR overexpression reflects saturation of adaptor-dependent recruitment of cargo into CCPs.

“That these kinetics were less affected by CD8/LDLR overexpression argues that reduced CME efficiency under these conditions reflects saturation of adaptor-dependent recruitment of cargo into CCPs.”

• AssumptionRests on an assumption: The efficiency of CME depends on cargo loading/concentration into CCPs.

The fluorescent tag did not interfere with Dab2 function.

“This expected localization of Dab2-mCherry suggests that the fluorescent tag did not interfere with its function.”

CCPs that take longer to pinch off have a higher ratio of clathrin:ARH than shorter-lived CCPs.

“Thus, CCPs that take longer to pinch off have a higher ratio of clathrin:ARH than shorter-lived CCPs.”

The presence or absence of a variable fraction of nonterminal events in different cell types could be explained by variable amounts of cargo and/or Dab2 at the plasma membrane.

“The presence or absence of a variable fraction of nonterminal events in different cell types could be explained by variable amounts of cargo and/or Dab2 at the plasma membrane.”

Similar GCCSs have been observed in other cell types upon Dab-2 overexpression, associated with integrins, another Dab2-specific cargo molecule.

“Similar GCCSs have been observed in other cell types upon Dab-2 overexpression, associated with integrins, another Dab2-specific cargo molecule (Chetrit et al., 2009).”

• cites Chetrit et al., 2009 sourceNot checked — no full text of the source

Recruitment of LDLR relies on an FXNPXY motif recognized by the adaptors Dab2 and ARH.

“recruitment of the LDLR relies on an FXNPXY motif (Davis et al., 1986) recognized by the adaptors Dab2 and ARH.”

• cites Davis et al., 1986 sourceNot checked — no full text of the source

GCCS formation is likely dependent on levels of expression of Dab2 and endogenous cargo.

“Hence, GCCS formation is likely dependent on levels of expression of Dab2 and endogenous cargo.”

• AssumptionRests on an assumption: Dab2 is recruited to CCPs and can function independently of AP2.

AP2 is required for ARH-mediated LDLR uptake.

“Altogether, these data clearly establish the requirement of AP2 for ARH-mediated LDLR uptake, which mirrors the requirements for ARH targeting to CCPs.”

Despite the dependence of ARH on AP2, ARH and AP2 compete for incorporation into CCPs.

“Together, these data indicate that despite the dependence of ARH on AP2, these adaptors nonetheless also compete for incorporation into CCPs”

Three CCP subpopulations exist: early abortive, late abortive, and productive CCPs.

“Recently, based on lifetime decomposition, we identified three CCP subpopulations, termed early abortive, late abortive, and productive CCPs (Loerke et al., 2009).”

• cites Loerke et al., 2009 sourceCheck pending

Increased efficiency of LDLR uptake upon ARH expression occurred through an increased efficiency of LDLR sorting/packaging into CCPs.

“Instead, it occurred through an increased efficiency of LDLR sorting/packaging into CCPs.”

The increase in CCP size by Dab2 and ARH might reflect the need to accommodate the very large ligands bound by LDLR and LRP (LDL particles, d∼22 nm and α2-macroglobulin, d∼18.5 nm).

“might reflect the need to accommodate the very large ligands bound by LDLR and LRP (Jones et al., 2003) (i.e., LDL particles, d∼22 nm and α2-macroglobulin, d∼18.5 nm; Krimbou et al., 1998), respectively.”

• cites Jones et al., 2003 sourceNot checked yet
• cites Krimbou et al., 1998 sourceCheck pending

Detection of nonterminal endocytic events in BSC1 cells expressing Dab2 suggests the existence of larger-than-normal CCPs or unresolvable clusters of CCPs even with endogenous cargo.

“Our ability to detect nonterminal endocytic events in BSC1 cells expressing Dab2 suggests the existence of some larger-than-normal CCPs or unresolvable clusters of CCPs, even in the presence of endogenous cargo levels.”

Both Dab2 and ARH are recruited into the same CCSs.

“Together, these results indicate that (i) Dab2 and ARH bind to the same cargo, (ii) both adaptors are recruited into the same CCSs”

Factors recruited to AP2 complexes through interactions with their appendage domains might provide input into the endocytic checkpoint.

“These data suggested that factors recruited to AP2 complexes through interactions with their appendage domains might provide input into the endocytic checkpoint.”

Additional factors required to stabilize productive CCPs become limiting during saturated CME.

“These effects suggest that additional factors, required to stabilize productive CCPs, become limiting during saturated CME.”

Under these conditions, larger CCPs assemble so that a residual clathrin patch remains after CCV budding.

“suggesting that under these conditions, larger CCPs assemble so that a residual clathrin patch remains after CCV budding.”

CD8/LDLR-dependent membrane recruitment of both ARH and AP2, because CCP density does not change, results in a cargo-dependent increase of adaptor protein content per CCP.

“Together, these results demonstrate a strong CD8/LDLR-dependent membrane recruitment of both ARH and AP2 that, because CCP density does not change, results in a cargo-dependent increase of the adaptor protein content per CCP.”

Despite partial functional redundancies and structural similarities, Dab2 and ARH exhibit fundamentally different, albeit cargo-dependent, interactions with CCPs.

“and (iv) despite their partial functional redundancies and structural similarities, Dab2 and ARH exhibit fundamentally different, albeit cargo-dependent, interactions with CCPs.”

Dab2 appears to sort cargo into CCPs independently of AP2.

“it appears to sort cargo into CCPs independently of AP2 (Garuti et al., 2005; Keyel et al., 2006; Maurer and Cooper, 2006).”

• cites Garuti et al., 2005 sourceNot checked yet
• cites Keyel et al., 2006 sourceNot checked — no full text of the source
• cites Maurer and Cooper, 2006 sourceNot checked — no full text of the source

Incorporation of transferrin receptor/AP2 into CCPs increases the efficiency of CCP maturation without affecting CCP density or CCP maturation rate.

“The incorporation of certain cargo/adaptor protein complexes (i.e., transferrin receptor/AP2) into CCPs increases the efficiency of CCP maturation without affecting CCP density or CCP maturation rate (Loerke et al., 2009; Mettlen et al., 2009).”

• cites Loerke et al., 2009 sourceCheck pending
• cites Mettlen et al., 2009 sourceCheck pending

At low levels of cargo, clathrin and/or AP2 binding by Dab2 are required for its recruitment to CCPs.

“These observations suggest that in the presence of low levels of cargo, clathrin and/or AP2 binding by Dab2 are required for its recruitment to CCPs.”

Dab2 and ARH are partially redundant for LDLR internalization.

“In these cells, Dab2 mediates LDLR internalization, suggesting that these adaptors are partially redundant (Maurer and Cooper, 2006).”

• cites Maurer and Cooper, 2006 sourceNot checked — no full text of the source

This recruitment of adaptor proteins had differential effects on CCP size and their dynamic behaviors.

“This recruitment of adaptor proteins had differential effects on CCP size and their dynamic behaviors.”

Nonterminal events are more frequent in HeLa cells, which express relatively high levels of Dab2 and cargo.

“This idea is further supported by the observation that nonterminal events are more frequent in HeLa cells (Ehrlich et al., 2004; Merrifield et al., 2005; Loerke et al., 2009), which express relatively high levels of Dab2 and cargo (Maurer and Cooper, 2006; unpublished data).”

• cites Ehrlich et al., 2004 sourceCheck pending
• cites Merrifield et al., 2005 sourceNot checked — no full text of the source
• cites Loerke et al., 2009 sourceCheck pending

Dab2 and ARH bind to the same cargo.

“Together, these results indicate that (i) Dab2 and ARH bind to the same cargo”

We identified a subset of endocytic accessory factors, including membrane curvature and coat-sensing proteins, whose siRNA knockdown altered the rate and extent of turnover of abortive CCPs.

“Subsequently, we identified a subset of endocytic accessory factors, including membrane curvature and coat-sensing proteins, whose siRNA knockdown altered the rate and extent of turnover of abortive CCPs (Mettlen et al., 2009).”

• cites Mettlen et al., 2009 sourceCheck pending

CCPs can take up multiple distinct receptors.

“Our data are consistent with the ability of CCPs to take up multiple distinct receptors.”

TfnR overexpression did not affect AP2 distribution.

“In contrast to TfnR overexpression, which did not affect AP2 distribution (Loerke et al., 2009)”

• cites Loerke et al., 2009 sourceCheck pending

The size of CCPs can be controlled by the ratio of Dab2/ARH.

“Together, these results indicate that (i) Dab2 and ARH bind to the same cargo, (ii) both adaptors are recruited into the same CCSs, (iii) the size of CCPs can be controlled by the ratio of Dab2/ARH”

Variations in adaptor/cargo content can account, in part, for the dynamic heterogeneity of CCPs.

“and that variations in adaptor/cargo content can account, in part, for the dynamic heterogeneity of CCPs.”

Overexpression of TfnR, a constitutively internalized AP2-dependent cargo, increased the efficiency of CCP maturation by increasing the contribution of productive CCPs at the expense of abortive species without affecting the rate of CCP maturation (lifetimes of productive CCPs).

“Previously, we showed that overexpression of TfnR, a constitutively internalized, AP2-dependent cargo, increased the efficiency of CCP maturation by increasing the contribution of productive CCPs at the expense of abortive species without affecting the rate of CCP maturation, i.e., the lifetimes of productive CCPs (Loerke et al., 2009).”

• cites Loerke et al., 2009 sourceCheck pending

ARH expression enhances CD8/LDLR uptake by increasing packaging, i.e., the number of CD8/LDLRs per CCP and/or the size of CCPs, rather than by enhancing either the efficiency or rate of CCP maturation.

“These data suggest that ARH expression enhances CD8/LDLR uptake by increasing packaging, i.e., the number of CD8/LDLRs per CCP and/or the size of CCPs, rather than by enhancing either the efficiency or rate of CCP maturation.”

ARH function is AP2 dependent.

“and its function is AP2 dependent (Maurer and Cooper, 2006).”

• cites Maurer and Cooper, 2006 sourceNot checked — no full text of the source

We previously identified an endocytic restriction/checkpoint and provided evidence that progression beyond the checkpoint (i.e., CCP maturation efficiency) was enhanced by TfnR overexpression in a manner dependent on AP2 adaptors.

“We previously identified an endocytic restriction/checkpoint and provided evidence that progression beyond the checkpoint (i.e., CCP maturation efficiency) was enhanced by TfnR overexpression in a manner dependent on AP2 adaptors (Loerke et al., 2009).”

• cites Loerke et al., 2009 sourceCheck pending

Even constitutively internalized cargo and their adaptors can regulate multiple aspects of CCP maturation.

“Together with our previous results (Loerke et al., 2009), we establish that even constitutively internalized cargo and their adaptors can regulate multiple aspects of CCP maturation”

• cites Loerke et al., 2009 sourceCheck pending