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Phosphoinositide species and filamentous actin formation mediate engulfment by senescent tumor cells [1]

['Wesley D. Frey', 'Tulane School Of Medicine', 'Department Of Biochemistry', 'Molecular Biology', 'New Orleans', 'Louisiana', 'United States Of America', 'Ashlyn Y. Anderson', 'Hyemin Lee', 'Julie B. Nguyen']

Date: 2022-11 

Similar dynamics were observed for 4226 mammary tumor cell line ( S1 Fig ). Taken together, the visualization of actin dynamics suggested 5 steps of the engulfment process: predator–prey contact, overtopping of the prey cell (ranging from approximately 10% to 90%), rounding of the prey cell, internalization, and degradation of the prey cell ( Fig 1L ).

To more quantitatively examine F-actin dynamics during maturation of the phagosome, we stained prey cells with pHrodo, a pH-sensitive dye that fluoresces brightly in the acidic environment of the lysosome. Thus, an increase in pHrodo (red) fluorescence intensity can only occur following maturation and acidification of the phagosome after the prey cell has been internalized by the predator cell. We found that predator F-actin was localized at a ring-like structure associated with the prey cell (as depicted in Fig 1I ) before engulfment and increased through engulfment, as pHrodo fluorescence increased ( Fig 1J ). Following the increase in pHrodo intensity as the prey cell was digested, the F-actin localized at the prey cell diminished sharply but was still maintained at a higher level than in the surrounding cytoplasm ( Fig 1J , Ring Intensity/Cytoplasm Intensity). The fluctuating predator F-actin levels localized to the prey cell contrasted with levels observed in nonengulfing areas of the membrane edge, where F-actin was present, but intensity never diminished over the engulfment time-course ( Fig 1J , Membrane Intensity/Cytoplasm Intensity). Fig 1K shows a time course of 1 representative LifeAct-GFP-expressing predator cell engulfing a pHrodo (red) stained prey cell. The predator “ring,” “membrane,” and “cytoplasm” areas used to calculate intensity are outlined and diagrammed in Fig 1I .

To more precisely localize F-actin in senescent predator cells during various stages of engulfment, we visualized LifeAct-GFP in axial cross sections, 3D volume view reconstructions, and videos. In early stages, we found F-actin concentrated across a broad area of contact with the prey cell, including advancing filapodial structures ( Fig 1C and S3 Video ). F-actin in the predator cell was tightly associated with the prey cell throughout the overtopping process ( Fig 1D and S4 Video ). At the time of apparent internalization, F-actin was highly concentrated in filopodial extensions present under and over the top of the prey cell ( Fig 1E and S5 Video ). Following engulfment and during early digestion as the prey cell repositioned within the predator, F-actin localized to a concentrated “belt-like” structure around the circumference of the prey cell ( Fig 1F–1H ).

Indicated “predator” MCF-7 cell lines were treated with 250 nM doxorubicin (DOXO) for 24 hours, washed, and plated on indicated not-treated (NT) MCF-7 “prey” cells, creating a “DOXO-NT” culture. Cells were imaged over days 4–8 as noted. ( A ) Volume view reconstruction of time course live-cell confocal imaging of a senescent LifeAct-GFP-MCF-7 cell during contact and partial overtopping of proliferating mCherry-MCF-7 cells on day 7 post-doxorubicin. Concentrated areas of F-actin are indicated by closed arrows, scale bar = 10 μm. ( B ) Volume view reconstruction of time course live-cell confocal imaging of a senescent LifeAct-GFP-MCF-7 cell following engulfment and through partial digestion of proliferating mCherry-MCF-7 cells. The top of the predator cell was removed above the 51.2% axial plane to visualize the internalized mCherry prey cell. Examples of concentrated F-actin are indicated by closed arrows, scale bar = 10 μm. ( C - E ) Axial slices along the Z-axis and volume view reconstruction of senescent LifeAct-GFP-MCF-7 cells during engulfment of a proliferating mCherry-MCF-7 cell. ( C ) shows contact and early overtopping, scale bar = 20 μm; ( D ) shows a mostly overtopped prey cell and a volume view with upper part of predator removed as in ( B ) scale bar = 20 μm. ( E ) shows a fully overtopped, engulfed cells with F-actin concentrated in lamellipodia and filipodia above and below the prey, scale bar = 25 μm. 3D volume view is shown from the bottom. ( F ) Volume view reconstruction of a senescent LifeAct-GFP-MCF-7 cell after fully engulfing a proliferating mCherry-MCF-7 cell. The top and side of the predator were removed along axial and sagittal planes to visualize engulfed cell, scale bar = 25 μm. ( G , H ) Confocal imaging with z-stack projections shown along right and bottom for senescent LifeAct-GFP-MCF-7 cells after fully engulfing proliferating mCherry-MCF-7 cells. At right of ( G ), volume views to visualize localization of the F-actin ring in the predator with an engulfed mCherry prey cell. Top right is top view, bottom right is side view, scale bar = 10 μm. At bottom of ( H ), axial slices displaying formation of smaller rings around digested portions of prey cell. Scale bar = 25 μm. ( I ) Schematic of a senescent LifeAct-GFP cell with 2 engulfed mCherry prey cells. Shown are example ROIs from the cell membrane, the cytoplasm, and the “ring” around the cell being engulfed. ( J ) Five individual LifeAct-GFP-MCF-7 predator cells were followed during engulfment of pHrodo red stained NT prey cells, and LifeAct-GFP intensity was measured at cytoplasm, nonengulfing membrane, and ring [as depicted in ( I )] and pHrodo intensity was measured (right). The left graph shows the ratio of LifeAct-GFP intensity in the ring:cytoplasm (light green) and membrane:cytoplasm over 7 hours for 5 cells; error bars represent SEM. The right graph shows pHrodo red intensity over the same time course. Underlying data can be found at S1 Data . ( K ) Time course images of 1 representative cell used to generate data for ( J ). Top: merged green/red channels with ROI used for pixel intensity measurements marked in yellow. Middle: merged green/red channels. Lower: green channel. ( L ) Model of predator cell F-actin localization during the 5 stages of engulfing a prey cell.

Over a time-course of 6 hours covering early stages of engulfment (contact, partial overtopping), confocal microscopy revealed F-actin concentrated in the predator cell at the points of contact with the prey cell, and at the leading edge of lamellar sheets during overtopping of the prey cell ( Fig 1A and S1A and S1B Video ). Following apparent internalization, F-actin concentrated and dissipated repeatedly around the prey cell(s) during the degradation process of approximately 7 hours ( Fig 1B and S2A and S2B Video ). F-actin formed structures reminiscent of those present intermittently on lysosomes and endosomes during propulsion through the cell or those associated with phagosomes and required for their mechanical deformation [ 31 , 32 ].

Actin reorganization is required for fundamental cell processes, including motility and phagocytosis. To monitor dynamics of filamentous (F)-actin formation during senescent cell engulfment, we expressed the biosensor LifeAct-GFP [ 30 ], a fusion protein that marks F-actin, in MCF-7 cells that were previously shown to engulf when senescent [ 6 , 27 ]. LifeAct-GFP cells were treated with doxorubicin (DOXO), and interactions with mCherry-expressing, not-treated (NT) cells were cocultured (“DOXO-NT”) and visualized by live-cell imaging and confocal microscopy.

Phospoinositide species regulate the entire process of whole cell engulfment by senescent cells

PI species interact with and modulate the activity of proteins that reorganize the actin cytoskeleton and are thus critical for the control of actin dynamics in macrophages during phagocytosis of various large targets [33–38].

We used specific biosensors to investigate involvement of different PI species in engulfment by senescent cells [39]. Live-cell time course imaging showed PLCD1-GFP [detecting PI(4,5)P2] and SidMx2-GFP [detecting PI(4)P] in the predator cell were closely associated with the prey cell during initial contact, at the overtopping stage, and through the point the predator cell became rounded and was detached from the substrate (Fig 2A and 2B). Both species dissipated in concentration as the digestion process ensued (Fig 2A and 2B). We observed in the predator cell a transient burst of highly concentrated 2xFYVE-GFP [detecting PI(3)P] enveloping the prey after overtopping that remained through rounding and internalization (Fig 2C). Imaging showed biosensors detecting PI(3,4)P2, PI(3,4,5)P3, and a mutant PLCD1-GFP biosensor that does not associate with any PI species (Negative Ctrl) were not concentrated or only diffusely associated with the engulfed cell (Fig 2D–2F), suggesting these species are not involved or are below the sensitivity of detection of their biosensors.

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TIFF original image Download: Fig 2. PI biosensors expressed in predator cells reveal PI(4,5)P2, PI(4)P, and PI(3)P, but not PI(3,4,5)P3 or PI(4)P, localize to the prey cells during engulfment. MCF-7 cell lines expressing biosensors that detect indicated PI species were treated with 250 nM doxorubicin for 24 hours, washed, and plated on untreated mCherry-MCF-7 “prey” cells. DOXO-NT cultures were imaged over days 3–8. Time course live-cell imaging of senescent MCF-7 cells that express (A) PLCD1-GFP marking PI(4,5)P2; (B) P4M-SidMx2-GFP marking PI(4)P; (C) 2xFYVE-GFP marking PI(3)P; (D) TAPP1-GFP marking PI(3,4)P2; (E) BTK-GFP marking PI(3,4,5)P3; (F) PLCD1(R40L)-GFP mutant that does not bind PI species (Negative Ctrl), throughout the entire process of engulfing mCherry-MCF-7 cells. Scale bar = 100 μm. https://doi.org/10.1371/journal.pbio.3001858.g002

To more precisely localize PI species in senescent predator cells during various stages of engulfment, we visualized the biosensors specific for PI(4,5)P2, PI(4)P, and PI(3)P, in axial cross sections, 3D volume view reconstructions, and videos. We found that PI(4,5)P2 was concentrated at the leading edge of the membrane as it contacted prey cells (Fig 3A, upper) and in the cell membrane as it advanced over and around the prey (Fig 3A, lower, and S6 Video). PI(4)P was similarly concentrated at contact early in engulfment (Fig 3B, upper, closed arrow), as the prey was partially overtopped (Fig 3B, upper, and S7 Video), and after complete overtopping and rounding of the prey cell (Fig 3B, lower). Fig 3C shows GFP fused to a mutant PI biosensor varies minimally in concentration within a predator and is only diffusely associated with prey cells during early through late stages of engulfment (Fig 3C). PI(4,5)P2 and PI(4)P both localized to the cell membrane and points of contact at the nonengulfing edges of the cell (Fig 3A and 3B). To determine if these PI species concentrate to a greater extent at sites of engulfment, we quantified the intensity at different regions of the engulfing cell (as depicted in Fig 1I) and compared to another membrane localizing protein that is not involved in engulfment, LYN11 [40,41]. We found the ratio of PLCD1-GFP to LYN11 was greater at the engulfing edge of the phagocytic cup than the nonengulfing edge or in the cytoplasm, suggesting PI(4,5)P2 was preferentially concentrated at sites of engulfment (Fig 3D). Similar results were observed for PI(4)P as detected by SidMx2-GFP (Fig 3E). Quantification over time lapse images showed PI(4,5)P2 dissipated at the prey cell before pHrodo fluorescence, suggesting PI(4,5)P2 was not involved in phagosome maturation post-engulfment (Fig 3F and 3G).

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TIFF original image Download: Fig 3. Confocal imaging shows precise localizations of PI(4,5)P2, PI(4)P, and PI(3)P in predator cells during engulfment. Axial slices along the Z-axis and volume view reconstructions of senescent MCF-7 cells, expressing different PI biosensors, during different stages of engulfment in DOXO-NT cultures. (A) PLCD1-GFP localization in predator cells during partial (top) and complete (bottom) overtopping of an mCherry-MCF-7 cell (top), scale bar = 20 μm (bottom), scale bar = 10 μm; (B) P4M-SidMx2-GFP localization in a predator cell during partial (top) and complete (bottom) overtopping of an mCherry-MCF-7 cell, scale bar = 10 μm. Closed arrows indicate examples of concentrated P4M-SidMx2-GFP localized to lamellipodia advancing over the prey cell. (C) Localization of PLCD1(R40L)-GFP mutant that does not bind PI species, during early (closed arrow), mid (dashed arrow), and late stage (open arrow) engulfments of mCherry-MCF-7 cells, scale bar = 10 μm. (D) MCF-7 cells coexpressing PLCD1-GFP and LYN11-mCherry (a control that localizes to membranes but is not involved in phagocytic processes) were imaged while engulfing NIR-MCF-7 cells. Left: representative images of 3 cells and the ROI used for quantitation indicated. Right: pixel intensities for the indicated ROI were determined and the ratio of intensity for PLCD1-GFP to LYN11-mCherry was calculated for 2–3 areas of the membrane edge actively contacting the prey cell (engulfing edge), 2–3 areas of the uninvolved membrane (nonengulfing edge), and 1 measurement per cell for the whole cytoplasm. Ratio calculations for 6 individual cells are shown as violin plots with mean and SEM indicated by red dashed line and black dashed line, respectively. Underlying data can be found at S1 Data. (E) Ratios as in (D) were calculated and shown for MCF-7 cells coexpressing SidMx2-GFP and LYN11-mCherry. Underlying data can be found at S1 Data. (F) Five PLCD1-GFP-MCF-7 predator cells were followed during engulfment of pHrodo red stained prey cells, and PLCD1-GFP intensity was measured at cytoplasm, membrane, and ring (as depicted in Fig 1I) and pHrodo intensity was measured (right). The left graph shows the ratio of PLCD1-GFP intensity of the ring:cytoplasm (light green) and membrane:cytoplasm over 12 hours for 5 cells; error bars represent SEM. The right graph shows pHrodo red intensity +/− SEM over the same time course. Underlying data can be found at S1 Data. (G) Time course images of 1 representative cell used to generate data for (F). Top: merged green/red channels with ROI used for pixel intensity measurements marked in yellow. Middle: merged green/red channels. Lower: green channel. Scale bar = 100 μm. In Fig 4D and 4E, two-way ANOVA was used for analysis, p-value < 0.05 = *, p-value < 0.01 = **, p-value < 0.001 = ***. https://doi.org/10.1371/journal.pbio.3001858.g003

Confocal imaging of 2xFYVE-GFP expressing cells revealed striking images of PI(3)P completely enveloping the prey after rounding (Fig 4A, upper and lower, S8 Video). Quantification of fluorescence intensity in different channels across the predator/prey showed predator cell PI(3)P concentrated at the prey cell far exceeding levels observed for LYN11-mCherry (Fig 4B and 4C). Time lapse imaging with pHrodo-labeled prey cells showed 2xFYVE-GFP was highly concentrated during the transient burst and remained enriched during digestion of the prey cell (Fig 4D and 4E).

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TIFF original image Download: Fig 4. Confocal imaging shows precise localization of PI(3)P in predator cells during engulfment. (A) Axial slices along the Z-axis and volume view reconstructions of a senescent MCF-7 cell expressing 2xFYVE-GFP and an mCherry expressing MCF-7 prey cell in a DOXO-NT culture. Top panel scale bar = 10 μm; bottom panel scale bar = 1 μm. (B, C) Representative image of a 2xFYVE-GFP cell engulfing a NIR-MCF-7 cell. Pixel intensity was determined across the distance of the yellow line and plotted in (B) for 5 individual cells. Scale bar = 60 μm. Underlying data can be found at S1 Data. (D) Five 2xFYVE-GFP-MCF-7 predator cells were followed during engulfment of pHrodo red stained prey cells, and 2xFYVE-GFP intensity was measured at cytoplasm, membrane, and ring (as depicted in Fig 1I) and pHrodo intensity was measured (right). The left graph shows the ratio of 2xFYVE-GFP intensity of the ring:cytoplasm (light green) over multiple hours surrounding pHrodo intensity increase for 5 cells. Error bars represent SEM. Underlying data can be found at S1 Data. (E) Representative images of one 2xFYVE-GFP-MCF-7 predator cell engulfing pHrodo-labeled MCF-7 prey used for Fig 4D. ROIs used for quantification are shown in top panel. Scale bar = 100 μm. (F) Time course of a 2xFYVE-GFP-MCF-7 cell engulfing an MCF-7-mCherry prey cell. Dashed arrows indicate predator/prey contact; closed arrows indicate the overtopped prey cell with 2xFYVE concentration; open arrows indicate the prey cell no longer overtopped mCherry prey cell. Scale bar = 50 μm. (G) Axial slices along the Z-axis and volume view reconstructions of senescent 2xFYVE-GFP-MCF-7 predator cells engulfing mCherry-MCF-7 prey cells. Leftmost image shows the bottom axial slice. Closed arrows indicate 2xFYVE-GFP concentration at the prey cell. Scale bar = 10 μm. https://doi.org/10.1371/journal.pbio.3001858.g004

In relatively rare instances, predator PI(3)P was observed concentrating at prey cells that were ultimately never engulfed (Fig 4F), suggesting PI(3)P localization was occurring before internalization. Indeed, confocal imaging showed PI(3)P tightly concentrated at prey cells that were clearly still attached to the substrate (Fig 4G). These data contrast to macrophages that localize PI(3)P only following phagocytic cup closure and during phagosome maturation [35,42–45], suggesting a novel role for PI(3)P generation in engulfment by senescent cells.

We observed accumulation of PI(4,5)P2, PI(4)P, PI(3)P at multiple stages of engulfment (Figs 2–4). To precisely discern differences in their localization and timing during engulfment, we coexpressed biosensors for PI(4,5)P2 and either PI(4)P (Fig 5A) or PI(3)P (Fig 5B) in GFP/mCherry combinations in the same predator cell, and imaged engulfment of near-infrared fluorescent protein (miRFP-713, “NIR”) expressing prey cells. Time-lapse imaging showed near perfect overlap of predator PI(4,5)P2 and PI(4)P during contact and through overtopping of prey cells (Fig 5A, NIR prey cells are blue). Confocal imaging in volume view, of axial slices, and in separated color channels also clearly showed colocalization of PI(4,5)P2 and PI(4)P in a predator cell at different stages of engulfing multiple prey cells (Fig 5C, NIR prey cells are pink). These data suggest similar or identical roles in engulfment and that phosphorylation of the 4 position on the inositol ring is significant.

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TIFF original image Download: Fig 5. PI(4,5)P2, PI(4)P colocalize at contact through overtopping of prey, then dissipate as PI(3)P localizes tightly around prey during later stages. (A) Time course live-cell imaging of a senescent MCF-7 cell that expresses PLCD1-mCherry and P4M-SidMx2-GFP, throughout the entire process of engulfing NIR-MCF-7 prey cells in a DOXO-NT culture. Closed arrows indicate examples PLCD1-mCherry and P4M-SidMx2-GFP colocalization during engulfment. (B) Time course live-cell imaging of a senescent MCF-7 cell that expresses PLCD1-mCherry and 2xFYVE-GFP, throughout the entire process of engulfing a NIR-MCF-7 cell. Closed arrows indicate localization of PLCD1-GFP, but not 2xFYVE-mCherry, at mid stage engulfment. Open arrows indicate 2xFYVE-mCherry localization, but not PLCD1-GFP, at late-stage engulfment. Scale bar = 100 μm for A+B. (C) Volume view reconstruction of a senescent MCF-7 cell expressing PLCD1-GFP and P4M-SidMx2-mCherry that is engulfing 3 NIR-MCF-7 cells. Middle panels show separate color channels; bottom panel shows axial planes from bottom to top. Closed arrows indicate examples PLCD1-mCherry and P4M-SidMx2-GFP colocalization during engulfment, scale bar = 10 μm. (D) Volume view reconstruction of a senescent MCF-7 cell expressing PLCD1-mCherry and 2xFYVE-GFP, which is engulfing 2 NIR-MCF-7 cells. Middle panels show separate color channels; bottom panels show axial planes from bottom to top. Closed arrows indicate localization of PLCD1-mCherry, but not 2xFYVE-GFP, at mid stage engulfment. Open arrows indicate 2xFYVE-mCherry localization, but not PLCD1-GFP, at an internalized cell, scale bar = 10 μm. https://doi.org/10.1371/journal.pbio.3001858.g005

Further imaging showed PI(4,5)P2 and PI(3)P were localized at entirely different stages of engulfment (Fig 5B and 5D). We found in the predator cell that PI(4,5)P2 was concentrated at the prey cell first, during contact and overtopping, but then dissipated as PI(3)P began to concentrate and localize. We observed very little overlap or colocalization during the transition (Fig 5B). This pattern of localization was evident in confocal imaging in a predator cell engulfing 2 NIR expressing prey cells (pink): one after internalization and one at overtopping. Imaging shows PI(4,5)P2 was concentrated and localized at the overtopped prey cell (Fig 5D) but not the internalized prey cell, while PI(3)P was concentrated and localized to the internalized, rounded prey cell (Fig 5D, green localization, open arrow). Similar PI species localizations were observed in 4226 cell line (S2 Fig). Dynamic localization of different PI species throughout the process of engulfment is depicted in S3 Fig.

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