Sorting and directed transport of membrane proteins during development of hippocampal neurons in culture

Abstract

Hippocampal neurons in culture develop morphological polarity in a sequential pattern; axons form before dendrites. Molecular differences, particularly those of membrane proteins, underlie the functional polarity of these domains, yet little is known about the temporal relationship between membrane protein polarization and morphological polarization. We took advantage of viral expression systems to determine when during development the polarization of membrane proteins arises. All markers were unpolarized in neurons before axonogenesis. In neurons with a morphologically distinguishable axon, even on the first day in culture, both axonal and dendritic proteins were polarized. The degree of polarization at these early stages was somewhat less than in mature cells and varied from cell to cell. The cellular mechanism responsible for the polarization of the dendritic marker protein transferrin receptor (TfR) in mature cells centers on directed transport to the dendritic domain. To examine the relationship between cell surface polarization and transport, we assessed the selectivity of transport by live cell imaging. TfR-green fluorescent protein-containing vesicles were already preferentially transported into dendrites at 2 days, the earliest time point we could measure. The selectivity of transport also varied somewhat among cells, and the amount of TfR-green fluorescent protein fluorescence on intracellular structures within the axon correlated with the amount of cell surface expression. This observation implies that selective microtubule-based transport is the primary mechanism that underlies the polarization of TfR on the cell surface. By 5 days in culture, the extent of polarization on the cell surface and the selectivity of transport reached mature levels.

Figure 1

Changes in the degree of polarization of axonal and dendritic markers during development. (a) In stage 2 neurons, axonal and dendritic markers are not segregated into different neurites. The micrographs illustrate a cell (phase, Left) from a 1-day-old culture 18 h after coinfection with adenoviruses encoding untagged versions of NgCAM (an axonal marker) and LDLR (a dendritic marker). At this stage, labeling of cell surface NgCAM (Center) and LDLR (Right) was primarily observed in the growth cones (arrow). Although the extent of staining varied among different neurites, both the axonal and dendritic markers tended to be concentrated in the same neurites. (Bar, 25 μm.) (b) In stage 3 neurons, axonal and dendritic markers have a complementary distribution, indicating their polarization to different neurites. The micrographs illustrate a stage 3 cell from a 1-day-old culture 18 h after coinfection with NgCAM- and LDLR-encoding adenoviruses. Labeling of cell surface NgCAM (Left) showed a strong polarization to the axon (arrows), including its growth cone, whereas staining of the short dendritic processes (arrowheads) was largely absent. In contrast, cell surface staining of LDLR (Right) was prominent in cell body and dendrites but nearly absent from the axon. (Bar, 25 μm.) (c) As a measure of polarization, we quantified the percentage of staining for each marker protein that was associated with the dendritic arbor. The dendritic proteins TfR and LDLR were already preferentially localized to the dendritic arbor on day 1, and their polarization increased to mature levels by day 5. Likewise, the axonal proteins NgCAM and L1 were preferentially excluded from the dendrites on day 1; their polarization was essentially complete by day 5. A pIgR construct whose sorting signal had been mutated (pIgR665–668) served to illustrate the distribution of an unsorted protein. The percentage of this protein associated with the dendritic membrane decreased slightly during development, paralleling the relative increase in size of the axonal arbor. TfR and pIgR685–668 were expressed with replication defective herpesviruses and LDLR and NgCAM with replication defective adenoviruses. L1 is an endogenous protein. Each point represents data from 10–20 cells examined 12–18 h after infection.

Figure 2

The polarization of TfR to the dendritic plasma membrane parallels the exclusion of TfR-containing carrier vesicles from the axon. TfR-GFP was expressed by using a defective herpesvirus. Cell surface TfR was assessed by staining living cells with an anti-TfR antibody, whereas GFP fluorescence served as a measure of all expressed TfR, including that associated with intracellular vesicles. (a and b) On day 2, the polarization of TfR varied somewhat from cell to cell. In some cells (a), surface staining for TfR (Center) was absent from the axon (arrows), which was paralleled by the absence of axonal TfR-GFP fluorescence associated with intracellular vesicles (Right). Staining in dendritic processes (arrowheads) was readily observed with both labels. In other cells (b), surface staining and TfR-GFP fluorescence were present in the distal axon (arrows) at a level comparable to that in the dendrites (arrowheads). The GFP fluorescence illustrates all TfR present in cells, including carrier vesicles in dendritic and axonal processes. (Bar, 20 μm.) (c) On the basis of a cell-by-cell comparison, there was a close correlation between the degree of polarization of cell surface TfR and TfR-GFP fluorescence. The total fluorescence in all dendritic processes was expressed as a percentage of the total fluorescence in all neurites including the axon. Cells in culture for 1 day were infected with replication-defective herpesvirus encoding TfR-GFP. After 18 h, living cells were stained with antibody to detect protein expression on the cell surface.

Figure 3

In stage 3 neurons, cell surface staining for NgCAM was restricted to the axon, whereas vesicles containing NgCAM-GFP were present in all processes (Left, phase contrast; Center, cell surface staining; Right, GFP; arrowheads denote dendrites). Cells in culture for 1 day were infected with replication-defective herpesvirus encoding NgCAM-GFP. After 18 h, living cells were stained with antibody to detect protein expression on the cell surface. (Bar, 20 μm.)

Figure 4

Comparison of the transport of TfR-GFP carrier vesicles in axons and dendrites of stage 3 cells. (a) A stage 3 neuron expressing TfR-GFP (Right, phase contrast; Left, GFP fluorescence); note the higher level of TfR-GFP fluorescence in the dendrites compared to the faint fluorescence in the proximal axon. Vesicle transport in this cell was recorded over a period of 30 sec, capturing images every 600 msec. Movies 1–4 of these data are published as supplemental data on the PNAS web site, www.pnas.org. (Bar, 20 μm.) (b) Vesicle transport in the proximal axon (Upper) and a representative dendrite (Lower). The topmost panel shows an enlarged view of the axonal segment (boxed in a). The path of each vesicle that moved in the anterograde direction or the retrograde direction during the 30-sec recording is shown in the two succeeding panels. Lower shows an enlarged view of one dendrite (boxed in a), followed by the path of each vesicle that moved in the anterograde and retrograde directions. Many more vesicles travel into the dendrite than the axon. To enable the visualization of faint vesicles in the axon, contrast was enhanced relative to the dendrite. (c) To quantify transport, recordings from TfR-GFP-expressing cells were analyzed by using kymographs, which show anterogradely moving vesicles as diagonal lines with positive slope, whereas retrogradely moving vesicles are represented by lines with negative slopes. This analysis revealed that there is extensive anterograde vesicle traffic into each dendrite but few transport events in the axon.

Figure 5

Changes in the amount of transport of TfR and NgCAM during development in culture. In the case of carrier vesicles labeled with TfR-GFP, the number of anterograde transport events in stage 2 cells (square), which lack an axon, is roughly comparable to the number of events seen in dendrites throughout development (open circles). In contrast, the number of TfR-GFP-containing vesicles entering the developing axon drops abruptly when the cells enter developmental stage 3 (filled circles). In the case of NgCAM, the number of anterograde transport events in dendrites remains constant during development, whereas the number of NgCAM-GFP-containing vesicles entering the axon increases gradually during development. Anterograde transport events were quantified by using kymograph analysis and normalized for the duration of the recording and the length of the neurite included in the image. The data for mature cells (>14 days in culture) were taken from ref. .