Membrane Trafficking in Disease: Mark A. McNiven

3D rotation of pancreatic cancer cells invading across a filter

Panc-1 cells invade across a coated filter overnight. It is easy to see the cells invading into the pores of the filter when viewed from the underside. Actin cytoskeleton (red), nuclei (blue), filter (white).

Live-cell microscopy of autolysosomal tubules being severed after washout of a dynamin inhibitor

A Hep3B hepatocyte expressing lysosomal-associated membrane protein 1 (LAMP1)-mCherry (mCh), a red fluorescent protein, was starved for two hours in Hanks' Balanced Salt Solution (HBSS)plus Ca2+/Mg2+ and a 10-millimolar concentration N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES). This was followed by a 30-minute treatment with 40 microns of dynasore (dynamin inhibitor). Movie starts after the dynasore is washed out. Movie is displayed at five frames per second and represents 10 minutes total elapsed time. Images were acquired every 20 seconds using epifluorescence microscopy to visualize the LAMP1-mCh tubules.

Source: Schulze, Ryan, et al. Lipid droplet breakdown requires dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes. Journal of Cell Biology, 2013; doi: 10.1083/jcb.201306140. Used under Creative Commons license. https://creativecommons.org/licenses/by-nc-sa/4.0

A pancreatic cancer cell degrades the substrate as it migrates

A BxPC3 pancreatic cancer cell expressing membrane-type 1 matrix metalloproteinase (MT1-MMP)-mCherry (mCh) (red) degrades the gelatin substrate (green) as it migrates. MT1-MMP-mCh can be seen at the leading edge of the cell as it moves. Images were acquired every five minutes for 20 hours using a confocal microscope. Playback is 10 frames per second.

Dynamin2 and actinin1 co-localize at the leading edge of a migrating pancreatic cancer cell.

A DanG pancreatic cancer cell expressing dynamin2-mCherry and actinin1-GFP (green fluorescent protein) migrating over the course of an hour. The cell shows several different lamellipodia during this time and migration is non-polarized. Images were acquired every 30 seconds for one hour. Playback is 10 frames per second.

Dynamin2 and actinin1 co-localize at the leading edge of a migrating pancreatic cancer cell

A DanG pancreatic cancer cell expressing dynamin2-mCherry and actinin1-green fluorescent protein (GFP) migrating over the course of an hour. The cell shows several different lamellipodia during this time, and migration is nonpolarized. Images were acquired every 30 seconds for one hour. Playback is 10 frames per second.

Circular dorsal ruffle formation in a hepatoma cell visualized with actinin1-GFP and super-resolution microscopy

A Hep3B hepatoma cell expressing the actin-binding protein actinin1-green fluorescent protein (GFP). A large circular dorsal ruffle forms and the dynamic actinin1-GFP can be seen. Images were acquired every minute for two hours and 12 minutes. Playback is 15 frames per second. Super-resolution imaging was performed with a Zeiss LSM 980 with Airyscan.

Macropinocytosis in hepatocyte visualized with dynamin2-GFP at 10 fps.

A clone9 rat hepatocyte expressing dynamin2-GFP (green fluorescent protein) can be seen undergoing macropinocytosis. The large dark macropinocytotic vesicles have a spot of dynamin 2 at the rear of the vesicle. Images were acquired every minute for one hour. Playback is 10 frames per second.

Macropinocytosis in hepatocyte visualized with dynamin2-GFP at 20 fps

A clone9 rat hepatocyte expressing dynamin2-green fluorescent protein (GFP) can be seen undergoing macropinocytosis. The large dark macropinocytotic vesicles have a spot of dynamin2 at the rear of the vesicle. Images were acquired every minute for one hour. Playback is 20 frames per second (fps).

Microlipophagy in a hepatocyte

Lipid (green) can be detected entering the lysosome (magenta) directly without engulfment of the lipid drop. Live-cell confocal imaging of a 4 micron by 4 micron region of a single alpha mouse liver 12 (AML12) hepatocyte cultured under basal growth conditions and treated with the orange-red fluorescent fatty acid BODIPY 558/568 C12 and LysoTracker Deep Red to label lipid drops and lysosomes in the green and magenta channels, respectively. Movie represents 24 minutes and 15 seconds of elapsed time with frames captured every five seconds. Playback is 20 frames per second.

Source: Schulze, Ryan J., et al. Direct lysosome-based autophagy of lipid droplets in hepatocytes. Proceedings of the National Academy of Sciences, 2020; doi.org/10.1073/pnas.2011442117. Used under Creative Commons license. https://creativecommons.org/licenses/by-nc-sa/4.0

Macrolipophagy in a hepatocyte

A lipid drop (green) is rapidly engulfed by a lysosome (magenta). Live-cell confocal imaging of a 7micron by 7 micron region of a single primary rat hepatocyte cultured under basal growth conditions and treated with BODIPY FL-C12 and tetramethylrhodamine (TMR)-dextran to label lipid drops and lysosomes in the green and magenta channels, respectively. Movie represents 33 minutes and 20 seconds of elapsed time with frames captured every 20 seconds. Playback is 20 frames per second.

Source: Schulze, Ryan J., et al. Direct lysosome-based autophagy of lipid droplets in hepatocytes. Proceedings of the National Academy of Sciences, 2020; doi.org/10.1073/pnas.2011442117. Used under Creative Commons license. https://creativecommons.org/licenses/by-nc-sa/4.0

MxB-mCh puncta dynamically form and disassemble over time

A Hep3B cell expressing myxovirus resistance 2 (MxB)-mCherry (mCh) imaged one frame every five minutes for over 16 hours and 40 minutes. Note the formation of MxB-mCh foci, which form and disassemble over the course of the video.

Source: Cao H, et al. The anti-viral dynamin family member MxB participates in mitochondrial integrity. Nature Communications. 2020; doi:10.1038/s41467-020-14727-w. Used under Creative Commons license. https://creativecommons.org/licenses/by-nc-sa/4.0

MxB-mCh puncta form and associate with mitochondria

A Hep3B hepatoma cell expressing myxovirus resistance 2 (MxB)-mCherry (mCh) (red) and mitochondrial matrix-localized (mito) green fluorescent protein (GFP) (white) imaged one frame every five minutes for over 16 hours and 40 minutes. The red MxB-mCh spots coalesce in the cytosol where they quickly associate with the mitochondria.

Source: Cao H, et al. The anti-viral dynamin family member MxB participates in mitochondrial integrity. Nature Communications. 2020; doi:10.1038/s41467-020-14727-w. Used under Creative Commons license. https://creativecommons.org/licenses/by-nc-sa/4.0

MxB-mCh puncta form and associate with mitochondria, another example

A second example of a Hep3B cell expressing myxovirus resistance 2 (MxB)-mCherry (mCh) (red) and mitochondrial matrix-localized (mito) green fluorescent protein (GFP) (white) imaged one frame every five minutes for over 16 hours and 40 minutes. The red MxB-mCh spots coalesce in the cytosol where they quickly associate with the mitochondria.

Source: Cao H, et al. The anti-viral dynamin family member MxB participates in mitochondrial integrity. Nature Communications. 2020; doi:10.1038/s41467-020-14727-w. Used under Creative Commons license. https://creativecommons.org/licenses/by-nc-sa/4.0

Pancreatic cancer cells sense a chemical gradient and migrate toward it

BxPC3 pancreatic cancer cells migrate toward a platelet-derived growth factor (PDGF) and serum gradient. BxPC3 cells were plated in a device containing media and 0.2% fetal bovine serum (FBS). On the left side of the field is a port where the media plus 10% serum and 30 nanograms per milliliter PDGF are added. The cells migrate toward the high concentration of serum with growth factor. Images acquired every two minutes for 17 hours. Playback is 30 frames per second.