Integrin-dependent trafficking of CD98hc reduces endocytic noise to maintain metabolic homeostasis

  1. F Sila Rizalar
  2. Hans Peter Grimm
  3. Angelique Augustin
  4. Jose L Garcia-Cordero
  5. Roberto Villaseñor

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    eLife Assessment

    This study offers an important set of findings on a previously unrecognised mechanism by which integrin-dependent trafficking, notably the recycling of CD98hc from endosomes to the plasma membrane, controls the abundance of a major amino acid transporter at the cell surface, with implications for a stable amino acid supply and metabolic homeostasis. The approach and results are convincing in showing the trafficking steps involved, although the experimental analysis is in places incomplete and would benefit from more rigorous approaches, deeper mechanistic characterisation, and stronger links to metabolic homeostasis. With these aspects strengthened, the work would be of interest to cell biologists working on trafficking and amino acid metabolism, as well as to the cancer cell metabolism community.

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Abstract

A stable supply of amino acids is essential for cellular homeostasis. CD98hc is an indispensable component of the LAT1 amino acid transporter. Here, we found an unexpected role for β1-integrin in amino acid homeostasis via the regulation of CD98hc trafficking. We identified Fast Endophilin-Mediated Endocytosis (FEME) as the primary internalization route for CD98hc in both osteosarcoma and brain endothelial cells. Endocytosis was followed by β1-integrin-dependent recycling, which diverted CD98hc from early endosomes. By combining mathematical modelling with high-content imaging, we demonstrate that this trafficking pathway exhibited emergent properties that filtered high-frequency endocytic events to stabilize CD98hc at the plasma membrane. Mutating CD98hc to disrupt its interaction with β1-integrin led to redistribution of the transporter into early endosomes, reduced intracellular amino acid levels and impaired growth under metabolic stress. We propose that regulation of transporter trafficking dynamics is a general mechanism to buffer endocytic noise and maintain metabolic homeostasis.

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  1. eLife

    eLife Assessment

    This study offers an important set of findings on a previously unrecognised mechanism by which integrin-dependent trafficking, notably the recycling of CD98hc from endosomes to the plasma membrane, controls the abundance of a major amino acid transporter at the cell surface, with implications for a stable amino acid supply and metabolic homeostasis. The approach and results are convincing in showing the trafficking steps involved, although the experimental analysis is in places incomplete and would benefit from more rigorous approaches, deeper mechanistic characterisation, and stronger links to metabolic homeostasis. With these aspects strengthened, the work would be of interest to cell biologists working on trafficking and amino acid metabolism, as well as to the cancer cell metabolism community.

  2. eLife

    Reviewer #1 (Public review):

    Summary:

    Dila Rizalar, Villaseñor and colleagues report the membrane trafficking steps undergone by CD98hc, a major component of the LAT1 amino acid transporter. They found that CD98hc entered cells through Fast Endophilin-Mediated Endocytosis (FEME) and quickly recycled back to the plasma membrane (PM) together with beta1-Integrin to mediate a stable supply of amino acids and support metabolic homeostasis.

    Strengths:

    The work was planned, executed and analysed to a high standard. A variety of cell models, techniques and assays were performed and combined with mathematical modeling. Appropriate analyses and statistical tests were used to support their conclusions.

    Overall, the evidence provided is convincing.

    Weaknesses:

    (1) The proposed endosomal recycling pathway(s) (positive for Rab9, Rab11 and Rab14) is/are not believed to support recycling fast enough (c. 10-20 min) to explain the very short timeframe (c. 1 min) reported by the authors. This apparent discrepancy should be clarified by the authors, ideally with small additional work.

    Notably, the fast Rab4-dependent recycling of TfR directly from Rab5 sorting endosomes has more consistent kinetics (c. 2-4 min, see the classic papers from the Mellman, Maxfield and Zerial labs) and should be investigated or at least discussed.

    (2) LAT1 (and thus possibly CD98hc) were previously reported to enter cells through Clathrin-mediated Endocytosis (CME) upon potent PKC activation (PMA) or entry into cell quiescence (Barthelemy and André, Sci Rep 2019 and Kahlhofer J et al. EMBO J 2025). Interestingly, instead of being recycled back to the PM, LAT1 was then routed to lysosomes for degradation.
    Does LAT1 co-traffic with CD98hc along the FEME - fast endosomal recycling loop identified by the authors?

    If so, the authors could discuss the physiological context in which the whole transporter shifts between the 2 routes (FEME-recycling in steady-state maintenance and CME-Lysosome upon acute signaling? There is a precedent in EGFR for such variation in endocytic fates upon signal strength).

    If not, the authors could discuss why the 2 chains would traffic differently (in such a scenario: CD98hc through the FEME-recycling arm and LAT1 through CME-Lysosome).

    (3) The study would benefit from mechanistic insights about the molecular details linking CD98hc to FEME.

    Specifically, identifying motif(s) and/or adaptor(s) binding to the main FEME adaptors (Endophilin A1, A2 or Bin1) would be a valuable improvement of the work and would provide a strategy to design a point mutant version of CD98hc that would be unlinked to FEME.

  3. eLife

    Reviewer #2 (Public review):

    Summary:

    In this manuscript, the authors demonstrated that the amino acid transporter CD98hc (SLC3A2) undergoes fast endophilin-mediated endocytosis (FEME), followed by rapid β1-integrin-dependent recycling back to the plasma membrane. The latter is essential to maintain CD98hc membrane levels and counteract bursts of endocytosis in both endothelial and osteosarcoma cells.

    Strengths:

    The authors used pharmacological inhibitors, siRNA-mediated downregulation and CRISPR approaches to identify the regulators of CD98hc trafficking in different cell systems, strongly supporting their conclusions. In addition, they complemented their experimental results with computational models, which further validate their conclusions

    Weaknesses:

    The mechanistic details of how the interaction between CD98hc and b1 integrin determines CD98hc recycling have not been explored in the current version of the manuscript. Moreover, despite the title mentioning metabolic homeostasis, there is limited insight into how changes in CD98hc recycling affect intracellular amino acid content and cell metabolism, and how this impacts cell growth.

  4. eLife

    Reviewer #3 (Public review):

    This paper from the Villasenor lab describes the results of experiments aimed at determining the role of the interaction of β1 integrin with CD98hc on the endo- exocytic trafficking of CD98hc, and its ability to function as an amino acid transporter. The paper demonstrates the following novel and interesting observations:

    (1) CD98hc is internalised via fast endophilin-mediated endocytosis (FEME).

    (2) A mutant of CD98hc that has been reported to oppose its interaction with β1 integrin (CD98hc ISM) alters CD98hc endo- exocytic trafficking to favour a situation in which CD98hc adopts a more endosomal distribution (than does wild-type CD98hc) following stimulation of FEME with a β-adrenergic agonist.

    (3) Driving CD98hc endocytosis with a β-adrenergic agonist reduces uptake of branched-chain amino acids (BCAAs); an effect which is more pronounced following expression of the β1 integrin binding-deficient CD98hc ISM mutant.

    General comments:

    The findings of this paper are, at their core, very interesting and important and most of the work is well-executed. I consider that the 3 conclusions that I have listed above are solid and interesting (although some additional controls, etc would help consolidate these findings (see below)). However, the authors have vastly overplayed their hand in attempting to use mathematical modelling approaches and (in my opinion) invoking unnecessary working hypotheses concerning endocytic noise for which they have little evidence. I feel that the paper needs to be restructured to simplify the route between the core findings and the interpretation, and to move the mathematical modelling and 'endocytic noise' analyses and hypotheses to the discussion.

    Specific points (in order of appearance):

    (1) I am puzzled by how the graphs in Figures 1E&F have been derived from primary data and am sceptical of their validity. The curves don't look like anything that has been derived from experimental data. I gather that the curves in Figures 1E&F were generated by the equations described in the 'modelling CD98hc internalisation kinetics..'methods section, but I really cannot see how. Specifically, how can measurements of steady-state plasma membrane (PM) and endosomal pools of CD98 possibly allow the derivation of the APM and ATE operands in the equations in the 'modelling CD98hc internalisation kinetics..' methods section? Moreover, I don't see how this could be achieved in a way that could demonstrate a statistically significant difference in the trafficking of TfnR and CD98hc. The authors cite their previous 2017 Cell Reports paper to support this figure. However, the data in this paper are clearly experimental data (therefore understandable and clear), and the analysis in Vallasenor et al. (2017) does not resemble what has been performed to generate Figures 1E&F. Can the authors please properly clarify and justify this figure?

    (2) Figures 1G&H. In the assay described in Figures 1G&H, it doesn't look like the CD98hc mAb is internalised in the control. And with the siRNAs that are purported to oppose CD98 internalisation (ARF6 and endophilin), it looks like CD98 has disappeared completely, rather than accumulating at the PM as one would expect if internalisation were blocked.

    (3) In Figure 1H, the authors state that knockdown of endophilin A1 and A2 reduced CD98hc endocytosis, but it doesn't really look like this to me and there are neither quants nor stats on this.

    (4) In Figure S1D, the conclusion that knockdown of dynamin or endophilin A1 and A2 decreases intracellular CD98hc-positive vesicles is not apparent to me, and these data need quantification and statistical analysis.

    (5) In Figure 1J, the conclusion that addition of dyngo4A reduces internalisation of anti-CD98hc is not statistically supported. Moreover, although β-adrenergic agonists are clearly boosting internalisation of CD98hc in U2OS cells (Figure S1J), this, despite the asterisks (stats), does not appear to be the case in ICE-BECs (Fig. 1J); indeed, the increase is marginal, and the inter-experimental variation is too great to support a statistically significant increase in internalised CD98hc in ICE-BECs.

    (6) The authors need to display representative images from experiments leading to the data in Figure 2B.

    (7) The data in Figure 2 do not 'show that CD98hc is sorted to recycling endosomes after FEME internalisation' in an integrin-dependent manner. For this, one would need to demonstrate localisation of the CD98hc-β1 integrin complex to Rab11 (or Rab14)-positive compartments and these data are missing.

    (8) I cannot find any details of the coordinates or construction of the CD98hc ISM mutant in this paper. I am assuming that this has been informed by the Ingber JCS paper from 2020, but the authors need to describe the construction and the provenance of their mutant. Also, importantly, the authors need to validate that this mutation opposes co-immunoprecipitation of β1 integrin and CD98hc. Or to use some other approach, such as FRET, to show this). OK, some of this has been performed by Ingber's group, but as it this is so important to the authors' conclusions, they need to confirm this. Also, it would be nice if the authors were to use their IP/proteomic approach to compare the interactomes of WT CD98hc and CD98 ISM.

    (9) I think that some of the callouts to Figures 3 A-F are not correct in the manuscript. For instance, at the top of the page of the results (no page numbers), the callout to the sentence '...expressing either mGL-CD98hc WT or ISM (Figure 3A-D)' is likely wrong, and this is confusing.

    (10) The data in Figures 3A,B,D&E do show that the CD98hc ISM mutant moves from the PM to endosomes more than does WT CD98hc following β-adrenergic stimulation. This is fine. However, I don't agree that the conclusions summarised in Figure 3C&F can be justified from these data. In fact, I do not believe it is necessary or justified to attempt to extrapolate these quite basic immunofluorescence data into an involved kinetic model. It would so improve the paper if these mathematical/kinetic extrapolations were moved to the discussion and presented as discursive points rather than hard-and-fast conclusions.

    (11) The data in Figure 4A do not appear to show anything further than those in Figures 3 3A,B,D&E. Indeed, in light of Figure 3 A,B,D&E one would expect the vesicular CD98hc to be increased by the ISM mutation, but I'm not convinced that the amplitude of these so-called oscillations is necessarily different between WT and ISM mutant of CD98hc. All the differences between WT and ISM in Figure 4A&B appear proportionally larger than they are because of the truncated (non-zero baseline) axes in these graphs; a linear axis such as the y-axes in Fig. 4A&B should begin at zero, surely?

    (12) In Figure 4D, it doesn't look at all as if the PM localisation of CD98hc and intracellular levels of BCAAs are similar between WT and ISM mutant of CD98hc - as the authors state in their results text.

    (13) Why do the authors need to invoke what appear to me to be rather far-fetched and poorly supported arguments around 'endocytic noise' when the data in Figures 3A,B,D&E seem to be sufficiently consistent with those in Figure 4E to support the assertion that altered trafficking of CD98hc influences BCAA uptake?. Surely, it would be sufficient to argue that a mutant transporter that assumes a state which is distributed more endosomally than at the PM would be less efficient in uptake of BCAAs. So why the noisy arguments? It just seems to me to undermine what is potentially quite a nice and interesting paper.

  5. eLife

    Reviewer #4 (Public review):

    Summary:

    Rizalar and colleagues present a study where they identified fast endophilin-mediated endocytosis (FEME) as the primary driver of CD98hc internalization in brain endothelial cells. They have characterized trafficking, intracellular localization, and amino acid transport under physiological conditions or metabolic stress using both experimental and mathematical modelling approaches.

    Strengths:

    The author presents an interesting study. The authors provide experimental and mathematical modeling to dissect integrin-dependent trafficking of CD98hc. They give an interesting role of integrin β1 different from adhesion.

    Weaknesses:

    (1) Overall, it is difficult to follow which cells were used for each experiment and the reasons. The authors should comment on the choice of osteosarcoma cells for the study, as the paper mainly discusses the choice of brain endothelial cells. Some key experiments in U2OS must be repeated in brain endothelial cells; targets identified in the siRNA screening should be tested in brain endothelial cells to increase the strength of conclusions.

    (2) Figure 1B is not clear in this disposition; it seems the same markers were used twice. Additionally, to improve clarity in understanding and reading, I suggest adding the cell line used in the figures.

    (3) In Figure S1A, it is difficult to conclude to the presence or absence of colocalization, zoom on ROI should be added as well as a quantification of the colocalization to conclude on the experiment.

    (4) Some context on intracellular sorting tubules can be added in the manuscript to improve clarity.

    (5) Figure S1C needs to be quantified to see if the mGL does not affect the expression of CD98hc; images could be added to support the absence of impact on cellular phenotype.

    (6) It could be worth measuring the localization of the surface pool of CD98hc to increase the strength of the conclusion, maybe by immunofluorescence colocalization between a cell membrane marker and CD98hc, or by pHluorine marker to differentiate cell surface from endosomes.

    (7) In Figure 1D, it seems that CD98hc have a different pattern in the upper panel compared to the lower one; it seems that vesicles are larger close to the membrane compared to deeper in the cytoplasm.

    (8) Some crucial controls are missing, mainly in siRNA experiments. For all siRNA experiments, efficiency and silencing must be shown by Western blot and quantified (Figure 1G, S1D-E).

    (9) In Figure S1D, the authors said "knockdown of ARF6 led to the accumulation of enlarged intracellular CD98hc positive structure". Size and number must be quantified to validate this assumption; I recommend adding an arrow to show structures for clarity. Same comment for the knockdown of dynamin and endophilin, the decrease of intracellular CD98hc positive structure need to be quantified.

    (10) In Figure S1G, only one or two cells are positive for GFP; the efficacy seems poor; in this sense, it's difficult to conclude whether they have an impact on internalization.

    (11) In Figure 2A, LAMP1 is significantly upregulated in the interactome; how do you interpret this, given that you show in Figure 1B that there is no colocalization.

    (12) Figure 2B: CD98hc colocalization with RAB subunits needs to be shown by microscopy in brain endothelial cells.

    (13) Overall, it is not clear if it's the integrin that is mutated for the binding domain or if it is the CD98hc binding domain that is mutated. If the integrin was not mutated, this type of experiment needs to be done to confirm the role of integrin. Additionally, immunoprecipitation needs to be done to show the absence or reduction of binding between CD98hc ISM and integrin.

    (14) Figure 3 was mainly done on U2OS cells; the integrin mutant needs to be done in at least one brain endothelial cell line. Do you observe the same results between the mutant integrin and KO integrin? The impact of b1 integrin on CD98hc could be tested using integrin b1 blocking antibodies.

    (15) The article focuses primarily on β1 integrin; could other integrins be involved? Given that integrins are often overexpressed in cancers, could this increase promote CD98hc internalization and trafficking? Adding this type of experiment could reinforce the conclusion and importance of integrin.

    (16) The proliferation of cells re-expressing CD98hc ISM under starvation or not shows a decrease in relative proliferation after 144 h. Could the authors comment on this? Is cell death happening?

    (17) The relevance of the results to the cancer field should be discussed in the discussion section, as osteosarcoma cells were used in the majority of experiments.

    (18) Integrins are mainly involved in adhesion; do the authors observe the same results between integrin and CD98hc if cells are cultivated on matrices like fibronectin?

    Please note, I have mainly reviewed the experimental part of the paper as I am not a specialist in mathematical modelling.

  6. Version published to 10.7554/elife.112816.1 on eLife
  7. Version published to 10.7554/elife.112816 on eLife
  8. Version published to 10.64898/2026.07.24.740607v1 on bioRxiv