The Musser Lab

  at the Texas A&M Health Science Center, College of Medicine


Please reference the appropriate paper(s) if you download our movies!

Notes:  Some of these Quicktime videos were made from 16-bit CCD camera data converted to 8-bit.  The Quicktime software on late model Macintosh computers may destroy the pixelation for video display.  If difficulties are encountered in attempts to observe the raw pixelated data, try using the RealPlayer software (Mac) or Quicktime on a PC.


From reference 30:
Tu, L., Fu, G., Zilman, A., and Musser, S. M. (2013) "Large Cargo Transport by Nuclear Pores:  Implications for the Spatial Organization of FG-Nucleoporins," EMBO J., 32:3220-3230.

Abortive Event.mov
Abortive Transport of M9-betaGal.  An M9-betaGal (500 kDa) import cargo molecule binds to a nuclear pore complex (NPC), but aborts transport.  The cargo was tagged with 8 Alexa647 dye molecules (red) and NPCs were labeled with GFP (green, EGPF-rPom121).  See Fig. 1C(top) of Reference 30 for more details.  Square pixels are ~240 nm on a side, each frame was acquired in 2 ms, and the playback speed is 5 frames per second.  Shown 100X SLOWER than real-time.

Entry Event.mov
Transport of M9-betaGal.  An M9-betaGal (500 kDa) import cargo molecule transports through a nuclear pore complex (NPC).  The cargo was tagged with 8 Alexa647 dye molecules (red) and NPCs were labeled with GFP (green, EGPF-rPom121).  See Fig. 1C(bottom) of Reference 30 for more details.  Square pixels are ~240 nm on a side, each frame was acquired in 2 ms, and the playback speed is 5 frames per second.  Shown 100X SLOWER than real-time.


From reference 29:
Sun, C., Fu, G., Ciziene, D., Stewart, M. and Musser, S. M. (2013) "Choreography of Importin-Alpha/CAS Complex Assembly and Disassembly at Nuclear Pores," Proc. Natl. Acad. Sci. USA, 110:E1584-E1593. PDF

Fig1A.mov
Association of Importin Alpha and muCAS Detected by smFRET during Nuclear Import.  <>Importin alpha was tagged with Alexa568 (donor fluorescence is yellow, left half of image) and muCAS was tagged with Alexa647 (acceptor fluorescence is red, right half of image).  The appearance of FRET reveals that an Imp-α/CAS complex was formed during nuclear import.  The bright-field background image underlay (blue) shows the position of the NE as a centrally located curve that bisects the images.  See Fig. 1A of Reference 29 for more details.  Square pixels are ~240 nm on a side, each frame was acquired in 1 ms, and the playback speed is 2 frames per second.  Shown 500X SLOWER than real-time.

Fig1D.mov
Dissociation of Importin Alpha and muCAS Detected by smFRET during Nuclear Export.  Importin alpha was tagged with Alexa568 (donor fluorescence is yellow, left half of image) and muCAS was tagged with Alexa647 (acceptor fluorescence is red, right half of image).  The disappearance of FRET reveals that an Imp-α/CAS complex was disassembled during nuclear export.  The bright-field background image underlay (blue) shows the position of the NE as a centrally located curve that bisects the images.  See Fig. 1D of Reference 29 for more details.  Square pixels are ~240 nm on a side, each frame was acquired in 1 ms, and the playback speed is 2 frames per second.  Shown 500X SLOWER than real-time.

Fig3B.mov
Transient Interaction of Importin Alpha and muCAS Detected by smFRET during Nuclear Import.  Importin alpha was tagged with Alexa568 (donor fluorescence is yellow, left half of image) and muCAS was tagged with Alexa647 (acceptor fluorescence is red, right half of image).  FRET was only observed for one frame  (1 ms), indicating a transient interaction.  The bright-field background image underlay (blue) shows the position of the NE as a centrally located curve that bisects the images.  See Fig. 3B of Reference 29 for more details.  Square pixels are ~240 nm on a side, each frame was acquired in 1 ms, and the playback speed is 2 frames per second.  Shown 500X SLOWER than real-time.

Fig3E.mov
Transient NLS-2xGFP/Importin Alpha/muCAS Complex Detected during Nuclear Import.  NLS-2xGFP was tagged with Alexa568 (donor fluorescence is yellow, left half of image) and muCAS was tagged with Alexa647 (acceptor fluorescence is red, right half of image).  NLS-2xGFP and muCAS are not expected to bind directly, but each bind to Importin Alpha.  Thus, the appearance of FRET (significantly weaker than for Fig1A.mov) indicates a trimeric complex of NLS-2xGFP, Importin Alpha, and muCAS.  The bright-field background image underlay (blue) shows the position of the NE as a centrally located curve that bisects the images.  See Fig. 3E of Reference 29 for more details.  Square pixels are ~240 nm on a side, each frame was acquired in 1 ms, and the playback speed is 2 frames per second.  Shown 500X SLOWER than real-time.


From reference 22:
Sun, C., Yang, W., Tu, L.-C. and Musser, S. M. (2008) "Single Molecule Measurements of Importin Alpha/Cargo Complex Dissociation at the Nuclear Pore," Proc. Natl. Acad. Sci. USA, 105:8613-8618. PDF Supplementary Material

Fig1A.mov
Dissociation of an Importin Alpha/Cargo Complex at a Nuclear Pore Complex (NPC).  Importin alpha was tagged with Alexa568 (donor dyes, yellow, left half of image) and NLS-2xGFP cargo was tagged with Alexa647 (acceptor dyes, pink right half of image).  Since only the donor dyes were excited, fluorescence in both channels (left and right halves of images) indicates fluorescence resonance energy transfer (FRET).  The presence of FRET indicates an intact complex.  The absence of FRET indicates that the complex dissociated.  The nuclear envelope (NE) is visible in the background bright field image (blue).  Loss of FRET occurs while the complex is at the NE, indicating that complex dissociation occurred at the NPC.  See Fig. 1A of reference 22 for more details. Square pixels are ~240 nm on a side, each frame was acquired in 2 ms, and the playback speed is 1 frame per second.  Shown 500X SLOWER than real-time.



From reference 20:

Hamai, C., Cremer, P. S., and Musser, S. M. (2007) "Single Giant Vesicle Rupture Events Reveal Multiple Mechanisms of Glass-Supported Bilayer Formation," Biophys. J. 92:1988-1999. PDF Supplementary Material

Fig2A.mov
Spontaneous GUV Rupture.  A single giant unilamellar vesicle (GUV) adsorbed to a glass coverslip spontaneously ruptures in ~10 ms.  The fluorescence emission arises from a fluorescent lipid (3% Texas Red-DHPE).  The initial GUV diameter was ~6 µm.  See Fig. 2A of reference 20 for more details.  Square pixels are ~150 nm on a side, each frame was acquired in 2 ms, and playback speed is 5 frames per second. Shown 100X SLOWER than real-time.

Fig6C.mov
Induced GUV Rupture.  A single GUV diffuses above a glass-adsorbed planar bilayer patch and eventually ruptures, presumably after diffusing to the glass surface and making contact with the edge of the planar bilayer patch.  See Fig. 6C of reference 20 for more details.  Square pixels are ~240 nm on a side, each frame was acquired in 2 ms, and playback speed is 10 frames per second. Shown 50X SLOWER than real-time.


From reference 19:
Yang, W., and Musser, S. M. (2006) "Nuclear Import Time and Transport Efficiency Depend on Importin Beta Concentration," J. Cell Biol. 174:951-961. PDF Supplementary Material

Video 1
A Nuclear Transport Event.  A single cargo molecule (NLS-2xGFP(4C) labeled with four Alexa647 dye molecules) transports through an NPC in a permeabilized HeLa cell nuclear envelope from the cytoplasmic compartment to the nuclear compartment.  See Fig. 1 of reference 19 for details of the conditions.  Square pixels are ~240 nm on a side, each frame was acquired in 2 ms, and playback speed is 10 frames per second. Shown 50X SLOWER than real-time. (red) Bright-field image intensity; (green) Alexa647 fluorescence intensity.

Video 2
An Abortive Nuclear Transport Event.  A single cargo molecule interacted with the same NPC as the cargo in Video 1.  Instead of transporting into the nuclear compartment, this cargo molecule returned to the cytoplasmic compartment.  Experimental and video conditions are identical to Video 1.


From reference 16:
Yang, W., Gelles, J. and Musser, S. M. (2004) "Imaging of Single-Molecule Translocation through Nuclear Pore Complexes" Proc. Natl. Acad. Sci. USA 101:12887-12892. PDF

Movie 1
An NPC Interaction Event.  A single Alexa-555-labeled NLS-2xGFP molecule interacts with the nuclear envelope (NE, dashed line). The single-molecule fluorescence (SMF) movie frames are overlaid onto a bright-field image.  The interaction event (white arrow) is defined as the appearance and disappearance of the red spot at the NE in the approximate center of the video. Nucleus and cytoplasm are identified.  Square pixels are ~225 nm on a side, each frame was acquired in 3.08 ms, and playback speed is 10 frames per second.  Shown 32X SLOWER than real-time. 

Movie 2
Two NPC Interaction Events.  The appearance of two nuclear envelope-binding events at the same location on the nuclear envelope are interpreted as two different cargo molecules interacting with the same nuclear pore complex (NPC).  Note that the NPCs do not diffuse within the nuclear envelope due to the nuclear lamins.  See Fig. 2b of reference 16 for signal quantification.  To reduce the file size, 789 ms of video was deleted between the two interaction events.  Video conditions and labeling are identical to Movie 1.  Shown 32X SLOWER than real-time.