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First published online 23 April 2003
doi: 10.1242/jcs.00526

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Rapid transport of neural intermediate filament protein

Brian T. Helfand*, Patty Loomis*, Miri Yoon and Robert D. Goldman{ddagger}

Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, 303 East Chicago Avenue, Ward 11-145, Chicago, IL 60611, USA



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  		Fig. 1. Peripherin particles, squiggles and longer IF. PC12 cells were fixed and processed for immunofluorescence at 0.5 hours (A-C), 2 hours (D-F) and 24 hours (G-I) after plating in DM. Peripherin particles and squiggles were apparent in regions between the nucleus and the cell surface between 0.5 and 2 hours (A-F; B and C are higher magnification views of the region denoted by the * in A; E and F are higher magnification views of the region indicated by the * in D). Longer IF were also present during this period. Growth cones and early neuritic processes, evident at ~2 hours, also contained particles and squiggles (D-F). After 24 hours, a large number of longer peripherin IF were present in most regions of differentiated cells, and particles and squiggles were most obvious in growth cones [G-I; the growth cone region (*) in G is seen at higher magnifcations in H and I]. Panels J-L show GFP-peripherin in a cell fixed at 2 hours in DM. The GFP fluorescence is observed directly in J-K, and peripherin is observed indirectly by anti-peripherin stained with lissamine-rhodamine-conjugated goat anti-rabbit in L. The * in J represents the growth cone region seen in K and L. Bar, 10 µm in A,D,G,J; Bar, 5 µm in B,E,H,K,L; Bar, 2 µm in C,F,I.

 


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  		Fig. 2. Peripherin is present in growth cones. A living PC12 cell expressing GFP-peripherin during the early stages of neurite outgrowth. This cell was observed at ~1-2 hours after plating in DM. Numerous GFP-peripherin particles and squiggles can be seen within the growth cone. A-C represents a single image from a time-lapse series. The image was captured by both phase-contrast and fluorescence microscopy to show the relationships between particles, squiggles, growth cones and filopodial extensions. The arrowheads indicate the positions of peripherin particles, some of which can also be detected with phase contrast. The particles and squiggles seen in the growth cone region are motile (see Movie 1 at jcs.biologists.org/supplemental). Bar, 5 µm in A-C. PC12 cells were plated in DM for 2 hours and then processed for platinum-replica immunogold TEM as described in Materials and Methods using rabbit anti-peripherin and gold-conjugated secondary antibodies. Ultrastructural observations (D-E) demonstrate that peripherin particles are present within the actin-rich growth cones and filopodia. In more proximal regions of growth cones, peripherin particles (indicated by clusters of 18 nm gold), as well as peripherin squiggles (most probably represented by linear arrays of gold; see arrows), are readily observed (F,G). E is a higher magnification view of area in the red box in D; F is a higher magnification view of the area in the green box in D; and G is a higher magnification view of the area in the blue box in F. Bar, 100 nm in D,E,G; Bar, 500 nm in F.

 


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  		Fig. 3. GFP-Peripherin is incorporated into endogenous IF. Analysis of IF-enriched cytoskeletal preparations of GFP-peripherin transfected PC12 cells. 2 µg/lane of an IF-enriched cytoskeletal extract from GFP-peripherin-transfected PC12 cells was separated by SDS-PAGE and immunoblotted with either anti-GFP (Lane 1) or anti-peripherin (Lane 2). The GFP antibody detects a single band at 84 kDa representing GFP-peripherin whereas the peripherin antibody detects both GFP-peripherin and the 57 kDa endogenous peripherin. Molecular weight standards are indicated at left.

 


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  		Fig. 4. Peripherin particles and squiggles move rapidly and bi-directionally. Analyses of the motile properties of particles and squiggles were made in GFP-peripherin-transfected cells in DM for periods of 2-4 hours (A-F) and at 48 hours (G,H). Observations were restricted to the peripheral regions of cell bodies and the central domain of growth cones at later time points, as both particles and squiggles were most obvious within these regions. A-E are derived from a time-lapse series (1 frame every 5 seconds) in the region of the cell body indicated by the box in the phase image (E). The particle marked * moved in a retrograde direction, and that marked with the arrowhead moved in an anterograde direction. F-H are diagrammatic representations of the trajectories of individual peripherin squiggles in the cell bodies (F,G) and in a growth cone (H). Black dots represent the beginning of squiggle tracks. A-D, Bar, 2 µm; E-H, Bar, 5 µm.

 


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  		Fig. 5. Particle and squiggle motility contribute to fluorescence recovery after photobleaching (FRAP). A bleach zone (areas denoted by red outlines) was made along the length of a neurite in a GFP-peripherin-expressing PC12 cell cultured in DM for 48 hours. Photobleaching required ~14 seconds (see A2, C2). Fluorescence recovery was subsequently monitored by determining the fluorescence intensity ratio (F.I.; see Materials and Methods) within this region by capturing images at ~60 seconds intervals for ~800 seconds. Using this ratio to determine the overall rate of fluorescence recovery, it was observed that the t1/2 (see A) for peripherin in this cell is ~400 seconds (also see A1-3, which represent the region prior to photobleaching, immediately following photobleaching at ~14 seconds and ~800 seconds after bleaching). The F.I. ratio was also determined for two subdivisions of the same region indicated by the large red box [B; B1-3 (subdivisions outlined in orange and green) at ~333, 385 and 448 seconds]. Using this more detailed analysis of recovery, transient peaks in the F.I. ratio were observed (see green and orange lines in B). These peaks were attributable to the rapid movements of bright squiggles and particles seen moving into and out of the bleach zone throughout the recovery period. In C, the same neurite has been separated into 10 subdivisions, including those depicted in B, each indicated by a different color on the graph (also see C1-3). This resulted in the complex series of peaks detected within the bleach zone during recovery. In addition, FRAP analysis was performed in a similar manner on another neurite of a PC12 cell grown in DM for 48 hours and then in DM containing 5 µg/ml colchicine for 30-45 minutes (D-1 to D-3). There was very little recovery up to 930 seconds after photobleaching (compare A with D). Interestingly, there was almost no fluctuation observed in the F.I. ratio and no particles or squiggles were observed to move within the bleach zone. Images E-H show GFP-peripherin particle movements through a photobleached area of the neurite shown in the phase image (I). Images were taken at 5 second intervals following photobleaching. The particle marked with an arrowhead moved in an anterograde direction at rates that ranged from 0.31-1.0 µm/second (also see Movie 2, available at jcs.biologists.org/supplemental). Diagrams of three trajectories of GFP-peripherin squiggles were made from another neurite (J). Black dots represent the beginning of squiggle tracks. Reversals of particles and squiggles were very infrequent within neurites of differentiated cells. E-H, Bar, 2 µm; I, J, Bars, 5 µm.

 


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  		Fig. 6. Peripherin association with microtubules and motors. Peripherin particles and squiggles are closely associated with microtubules (MT) in the growth cone of a PC12 cell in DM for 4 hours. (A-D) Double label immunofluorescence. (A) peripherin (red); (B) microtubules (green); (C) overlay. D is a magnified view of the peripheral region of the growth cone shown in C. (E) Overlay of a double-labeled immunofluorescence preparation showing the association (yellow) between peripherin particles and squiggles (red) and kinesin (green). (F) Overlay of double label immunofluorescence preparation showing the association (yellow) between peripherin (red) and dynein IC (green). Bars, 2 µm.

 


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  		Fig. 7. Individual peripherin structures can associate with both motors. GFP-peripherin-transfected PC12 cells were plated in DM for 4 hours and then processed for indirect immunofluorescence using anti-kinesin heavy chain (red) and dynein heavy chain (blue). It was determined that the majority of particles and squiggles associate with both kinesin and dynein (A,B,C, see asterisk in F). Some of these peripherin structures associate only with dynein (A,C,E, see arrow in F), others associate only with kinesin (A,B,D, see filled arrowhead in F), and a small percentage do not appear to associate with either motor (see open arrowhead). Bar, 5 µm.

 


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  		Fig. 8. Ultrastructural analysis of peripherin particles within growth cones. PC12 cells plated in DM for 4 hours were processed for platinum replica immunogold electron microscopy using a rabbit polyclonal peripherin antibody, a mouse monoclonal kinesin heavy chain antibody and/or a mouse monoclonal dynein intermediate chain antibody. Secondary antibodies were 10 nm gold-conjugated anti-rabbit and 18 nm gold-conjugated anti-mouse antibodies. In the central domain of the growth cone, many particles associated with MT and their associated motors. A-C demonstrate kinesin and peripherin association. C is a color overlay showing peripherin (green), kinesin (pink) and MT (yellow). D-F show an association between peripherin and dynein. F is a color overlay showing peripherin (green), dynein (pink) and MT (yellow). A and D, Bar, 600 nm; B,C,E,F, Bar, 100 nm.

 


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  		Fig. 9. IF-enriched cytoskeletons contain motor subunits. Lane `IF' is a Coomassie stain showing that peripherin (P) is the major protein present in IF-enriched cytoskeletal preparations. Immunoblot analysis of these same preparations shows that kinesin and many of the components of dynein and dynactin are present. MW indicates molecular weight standards. DIC, dynein intermediate chain; DLIC1 /2, dynein light intermediate chain isoforms 1 and 2; DHC, dynein heavy chain; p50, dynamitin; Arp-1, actin related protein-1; KHC, kinesin heavy chain.

 


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  		Fig. 10. Disruption of motors alters the distribution of peripherin. PC12 cells grown on locator coverslips in DM for 48 hours were microinjected with kinesin heavy chain antibody (C,D) or as a control, with non-immune serum (A,B), and then processed for immunofluorescence using peripherin antibody at 0.5-4 hours post-injection. Control cells displayed normal peripherin networks (A). In cells injected with kinesin antibody the peripherin was almost exclusively located in the cell body (C). B and D are phase images of the same injected cells.PC12 cells were also transfected with myc-dynamitin cDNA (G) or mock transfected (E) and processed for immunofluorescence with peripherin and c-myc (data not shown) antibodies, 48 hours post-transfection. Dynamitin-expressing cells showed peripherin staining almost exclusively in the peripheral regions of the cell body and distal regions of neurites (G). Mock-transfected cells displayed peripherin networks that were typical of well-differentiated cells. Phase contrast (F,H). Bars, 10 µm.

 

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© The Company of Biologists Ltd 2003