Golgi localized Arl15 regulates cargo transport, cell adhesion and motility

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Arf-like GTPases (Arls) regulate membrane trafficking and cytoskeletal organization. Genetic studies predicted a role for Arl15 in type-2 diabetes, insulin resistance, adiposity, and rheumatoid arthritis. Recent studies indicate a possible role for Arl15 in multiple physiological processes, including magnesium homeostasis. However, the molecular function of Arl15 is poorly defined. We evaluated the role of Arl15 in vesicular transport using techniques to quantify cargo trafficking, to mechanobiology. Fluorescence microscopy of stably expressing Arl15-GFP HeLa cells showed its localization to the Golgi and cell surface, including filopodia, and a cohort to recycling endosomes. The dissociation of Golgi, using small molecular inhibitors or the expression of Arf1 dominant-negative mutant, completely mislocalized Arl15 to the cytosol. Interestingly, site-directed mutagenesis analysis identified a novel V80A mutation in the GTP-binding domain that turns Arl15 into a dominant-negative form with reduced number of filopodia. Depletion of Arl15 in HeLa cells caused mislocalization of cargo, such as caveolin-2 and STX6, from the Golgi. Arl15 knockdown cells displayed reduced filopodial number, altered focal adhesion kinase organization, and enhanced soluble and receptor-mediated cargo uptake without affecting the TfR recycling. Arl15 knockdown decreased cell migration and enhanced cell spreading and adhesion strength. Traction force microscopy experiments revealed that Arl15 depleted cells exert higher tractions and generate multiple focal adhesion points during the initial phase of cell adhesion as compared to control cells. Collectively, these studies demonstrated a functional role for Arl15 in the Golgi, which includes regulating cargo transport to organize membrane domains at the cell surface.

Key points

  • Arl15 primarily localizes to Golgi and plasma membrane, including filopodia

  • Membrane localization of Arl15 is dependent on Golgi integrity or Arf1 activation

  • Arl15 knockdown mislocalizes STX6-dependent Golgi localized cargo required for cell surface organization and reduces the filopodial number

  • Arl15 is involved in cell spreading, adhesion, and migration

  • Article activity feed

    1. Review coordinated via ASAPbio’s crowd preprint review

      This review reflects comments and contributions by Vasihvani Ananthanarayanan, Sam Lord, Rinalda Proko, Luciana Gallo, Sónia Gomes Pereira, Asli Sadli, Mugda Sathe, Parijat Sil. Review synthesized by Iratxe Puebla.

      The preprint studies the molecular function of Arl15, a member of the Arf-like GTPases (Arls) group, which has been linked to magnesium homeostasis. The manuscript reports that Arl15 localizes in the Golgi and plasma membrane, including filopodia. The dissociation of Golgi or the expression of Arf1 dominant-negative mutant leads to a mislocalization of Arl15 to the cytosol. Knocking down Arl15 results in reduced filopodial number, altered focal adhesion kinase organization, and enhanced cargo uptake. Arl15 knockdown decreases cell migration and enhances cell spreading and adhesion strength. The findings point to a functional role for Arl15 in the Golgi.

      General comments

      • Figures 1,2, 3 - The images display one representative example, recommend providing quantification (e.g. PCC/Manders) across several biological replicates, as well as information on the type of images reported, single slice, max Z projection etc.
      • For the bar plots, the paper reports the number of cells as well as the number of times the experiment was repeated, which is excellent. However, it is unclear whether the SEM error bars and p-values were calculated based on the number of repeats (correct) or based on the number of cells (incorrect). Can clarification be provided for this point See https://doi.org/10.1083/jcb.202001064 and https://doi.org/10.1371/journal.pbio.2005282.
      • Throughout the paper there are several references to ‘data not shown’ - please report the data for those items.

      Specific comments

      Introduction, first paragraph - Suggest shortening the paragraph, particularly regarding the description of the different Arls and their relationship/correlation with all diseases.

      ‘These results show that similar to HeLa cells, Arl15-GFP localizes to PM along with filopodia and Golgi in all mammalian cell types’ - Suggest revising the fragment to ‘all the mammalian cell types tested in the study’, to avoid generalizing to every mammalian cell type.

      the localization of Arl15-GFP to PM however remained unchanged as compared to DMSO treated cells (Fig. 2A).’ - Fig 2A only compares mCherry-UtrCH against Arl15-GFP. To support this claim, Arl15-GFP would need to be compared to WGA-AF, as in Figure 1, and their colocalization quantified to confirm that it remained unchanged.

      ‘We treated mCherry-UtrCH expressing HeLa:Arl15-GFP stable cells with a small molecular inhibitor of Rac1 (CAS 1177865-17-6) or Cdc42 (ML141)’ - Please report the concentration of both inhibitors.

      ‘Overall, these studies indicate that neither actin depolymerization nor the key regulatory molecules of filopodia/lamellipodia affect the localization of Arl15 to PM/Golgi.’ - The visualization reports Arl15-GFP v mCherry-UtrCH, to support the claim please check against WGA/GM130 as in Figure 1. Also, Figure 2c Arl15 for FAK inhibitor looks different from the DMSO control, recommend confirmation with WGA staining. Can also some explanation be provided for the fact that the Arl-15 in Figure 2A and 2C DMSO looks quite different from 2B and 2D despite the stable cell line with uniform expression?

      ‘which mislocalized Golgi pool of Arl15 without affecting its PM localization (Fig. 2D)’ - There does not seem to be a marked difference in Arl15-GFP's intracelluar localisation in cells with and without microtubules, and the PM signal appears slightly reduced in the Nocodazole-treated cells. Is it possible to please quantify the localisation?

      Figure 2 -The quality of the images from panels B and D looks very different from those of A and C. Can some clarification be provided, were the same microscope, camera, and settings used?

      Figure 3 - It would be good to mention the role of brefeldin A as an ATPase inhibitor to provide context for why it is being used.

      ‘Surprisingly, Arl15-GFP localized to the cytosol as similar to Arf1-GFP in GM130 dispersed cells that are indicative of brefeldin A treatment in HeLa cells (Fig. 3A).’ - It may be worth clarifying the reference to a surprising result, considering the nocadozol results would this result not be expected? It may also be worth providing some comments about the possible PM localisation difference when Golgi is disrupted with nocadozol vs BrefeldinA/golgicide A. It seems that the PM localisation is also affected in the BrefeldinA/golgicideA treatments.

      Figure 3A ‘Cells were treated with DMSO (as control), brefeldin A or golgicide A for 24 h followed fixation’ - Please comment on the 24-hour period, BFA would be expected to work in minutes timescale: https://rupress.org/jcb/article-pdf/118/4/813/1063231/813.pdf

      Supplementary Fig 2A - The blots for Arl15 endogenous are very different between S2A and S2B. Also a 40% knockdown of Arf1 decreases the level of Arl15 by 17%. Can some comments be provided on the significance of this decrease.

      Figure 4 - Is the SEM over 3 independent experiments or total number of cells from the three experiments? What was the criteria used to define a structure as filopodia?

      ‘However, we continued with Arl15V80A,A86L,E122K cytosolic mutant to study the functionality of Arl15 in HeLa cells’ - It may be worth specifying the reason to use the V80A,A86L,E122K form instead of the more simple V80A alone?

      ‘To test whether the mislocalized Cav-2 and STX6 are targeted to lysosomes in siArl15 cells’ - Please comment on why colocalisation with lysotracker or lamp1 positive structures was not examined instead of treating the cells with bafilomycin A1? Note that bafilomycin A1 also inhibits retrograde membrane traffic at the ER–Golgi boundary: https://www.molbiolcell.org/doi/10.1091/mbc.9.12.3561?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed

      Figure 5 - Please clarify whether the quantification of images was done on images taken from the same microscope? Also, suggest arranging the figures in a way that the quantification and images are not so far apart from each other.

      Figure 5D - It is unclear how the western blot of EGFR showing total EGFR is indicative of what happened to its trafficking, this appears to be in contrast to the increase in transferrin uptake data. Recommend normalizing the transferrin uptake to surface transferrin levels as one can have higher uptake simply because there is more transferrin receptor instead of actual changes in trafficking rates.

      ‘Nevertheless, the reason for the partial loss of STX6 and caveolin-2 localization from Golgi in the ASAP1/2 knockdown cells requires investigation’ - Text earlier mentioned "However, we have not observed any significant change in Arl15 and its dependent cargo (caveolin-2 and STX6) localization to Golgi in siASAP1/2 cells " and there does not appear to be any difference in the siASAP1 or siASAP2 on Fig 6. However, in Figure S3 there is a slight reduction in the intensity. Can this be clarified?

      Methods ‘Post chase, cells were washed with 1X PBS, fixed with 3% formaldehyde…’ - Please report for how long and at which temperature the fixation step was completed.