INF2-mediated actin polymerization at ER-organelle contacts regulates organelle size and movement

Abstract

Proper regulation of organelle dynamics is critical for cellular function, but the mechanisms coordinating multiple organelles remain poorly understood. Here we show that actin polymerization mediated by the endoplasmic reticulum (ER)-anchored formin INF2 acts as a master regulator of organelle morphology and movement. Using high-resolution imaging, we demonstrate that INF2-polymerized actin filaments assemble at ER contact sites on mitochondria, endosomes, and lysosomes just prior to their fission. Genetic manipulation of INF2 activity alters the size, shape and motility of all three organelles. Our findings reveal a conserved mechanism by which the ER uses actin polymerization to control diverse organelles, with implications for understanding organelle dysfunction in neurodegenerative and other diseases. This work establishes INF2-mediated actin assembly as a central coordinator of organelle dynamics and inter-organelle communication.

Figure 1:. ER-associated actin accumulates at endosomal, lysosomal, and mitochondrial fission sites prior to organelle division.

U2OS cells were co-transfected with plasmids directing expression of fluorescently-tagged organelle markers and fluorescently-tagged, ER-targeted actin nanobodies (“AC-ER”) and timelapse videos were collected. A. Endosomes are labeled with Rab5-mCherry, lysosomes were labeled with LAMP1-mCherry, and mitochondria were labeled with MitoTracker Deep Red (magenta). AC-ER GFP is shown in green. Example organelles are shown at different time points prior to and immediately after the fission event. Arrowheads indicate the fission sites. White dotted lines have been drawn around the “daughter organelles” resulting from the fission event in the bottom panel for clarity. To aid visualization, line scans were drawn over the fission sites and the surrounding regions. The resulting pixel intensities associated with the line scans are shown. Yellow lines indicate the region where the line scan was drawn (shifted so as not to block visualization of the organelle). Scale bars are 1μm. B. Graph comparing the frequency of observed presence/absence of AC-ER at organelle fission events (green bars) versus the possibility of AC-ER being at fission events by chance (purple bars). Actual observed AC-ER at fission events was determined by manually scoring fission events in a blinded fashion, followed by looking for AC-ER signal at the identified fission events. By chance values were determined by calculating the percent of organelle signal overlapped by AC-ER signal (described in greater detail Supp. Fig 1D and Methods). Observed versus by chance values were statistically compared via Fisher’s exact test. C. Tabular breakdown of the n-values and p-values associated with AC-ER fission event analysis. For each organelle + AC-ER combination the n-values corresponding to the number of cells analyzed as well as the number of fission events scored are displayed. The calculated percent overlap (corresponding to the purple bars in B) are shown in the second column. These values were used to calculate the “expected” percentage of fission events where AC-ER would be present if due to chance. The observed and expected percentages were compared via Fisher’s exact test and the resulting p-values are displayed in the last column (corresponding to the p-values in B). All experiments were performed with N=3 biological replicates.

Figure 2:. Loss of INF2 causes organelle enlargement and elongation.

Experiments were carried out in wild-type U2OS cells (“Control”) and U2OS cells where the ER-anchored isoform of INF2 has been knocked-out (“INF2-ER KO”). Cells were imaged live following transfection with Rab5-mCherry to label endosomes, LAMP1-mCherry to label lysosomes, or staining with MitoTracker Deep Red to label mitochondria. The leftmost column displays representative images of each organelle in Control U2OS cells. The following column shows each organelle in INF2-ER KO cells. The following column displays INF2-ER KO cells that have been co-transfected with INF2-ER GFP. The following column displays INF2-ER KO cells co-transfected with INF2-ER[K792A] GFP. The following column displays INF2-ER KO cells co-transfected with INF2-Cyto GFP. The last column displays quantification of individual organelle area for each condition. Black dots show the average per cell and bars indicate the average per condition. Error bars show standard deviation. N = 12 cells per condition for endosomes and lysosomes and 24 cells per condition for mitochondria. For all graphs, the magnitude of the change and p-value compared to control is shown. An up arrow indicates an increase and a down arrow indicates a decrease. For statistical comparisons **** indicates p-value≤0.0001, *** indicates p-values≤0.001, ** indicates p-value≤0.01, * indicates p-value≤0.05, and ns indicates p-value>0.05. Conditions were compared via ordinary one-way ANOVA. All experiments were performed with N=3 biological replicates.

Figure 3:. Loss of INF2 increases organelle mobility.

Organelles were labeled in wild-type U2OS cells (control) and INF2-ER KO U2OS cells using Rab5-mCherry (endosomes), LAMP1-mCherry (lysosomes), or MitoTracker Deep Red (mitochondria) and their movement was tracked during 5-minute timelapses using Imaris software. To aid in visualization of organelle movement, tracks marking the trajectories of each organelle in representative cells are shown. These tracks are color-coded according to Imaris’ calculated “track displacement distance”. This is the measured distance between the organelle’s location at the beginning and its location at the end of the timelapse. Thus, more red tracks indicate organelles that cover more distance, and therefore, move more, while more blue tracks indicate organelles that move less. The first column shows tracks generated from endosome, lysosome, and mitochondrial movement in the Control condition. The following column displays organelle tracks from the INF2-ER KO condition. The following column displays tracks from INF2-ER KO cells co-transfected with INF2-ER GFP. The following column displays tracks from INF2-ER KO cells co-transfected with INF2-ER[K792A] GFP. The following column shows tracks from INF2-ER KO cells co-transfected with INF2-Cyto GFP. The last column shows quantification of organelle movement based on Imaris organelle tracking. To correct for tracks that do not last the entire duration of the timelapse due to organelles leaving/entering the frame or going in/out of focus, the “Mean Straight Velocity” of each organelle was calculated by dividing the track displacement distance by the track duration. N = 12 cells per condition for endosomes and lysosomes and 24 cells per condition for mitochondria. For all graphs, black dots show the average per cell and bars indicate the average per condition. Error bars show standard deviation. The magnitude of the change and p-value compared to control is shown. An up arrow indicates an increase and a down arrow indicates a decrease. For statistical comparisons **** indicates p-value≤0.0001, *** indicates p-values≤0.001, ** indicates p-value≤0.01, * indicates p-value≤0.05, and ns indicates p-value>0.05. Conditions were compared via ordinary one-way ANOVA. All experiments were performed with N=3 biological replicates.