Motor Switch from KIF4 to KIF5 Induces a Selective Reduction in Anterograde Velocity of Fluorescent Cellular Prion Protein in Neurites

Copyright: © 2013 Kato H, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
The prion protein (PrP) consists of two isoforms, a host-encoded cellular isoform (PrP C ) and an abnormal protease-resistant pathogenic isoform (PrP Sc ); the latter is the causative agent of prion diseases. PrP Sc stimulates the conversion of PrP C into nascent PrP Sc , and accumulation of PrP Sc leads to central nervous system dysfunction and neuronal degeneration [1]. Initial degradation of PrP C involves the cleavage of the NH 2 -terminal fragment to produce a 17-kD COOHterminal polypeptide, which can be recovered in a Triton X-100 insoluble fraction [2][3][4]. The NH 2 -terminal fragment itself functions as a putative targeting element [5,6] and is essential for both movement to the plasma membrane and modulation of endocytosis [7]. A NH 2 -terminal GFP-tagged version of PrP C (GFP-PrP C ) was found to anchor properly at the cell surface, and its distribution pattern was similar to that of endogenous PrP C [8][9][10][11] and a COOH-terminal tagged version (PrP C -GFP) [12].
Knowledge of the physiological role of cellular prion protein is important for the understanding of prion disease; however, despite many efforts, the exact role of cellar prion protein remains unclear. For this reason, we have investigated cellular prion protein by transient transfection of fluorescent PrP C . We previously demonstrated a microtubule (MT)-associated intracellular localization and movement of the NH 2 -terminal fragment of fluorescent PrP C [13] in mouse neuroblastoma neuro2a (N2a) cells, which are known to be infectable with PrP Sc [14], and in HpL3-4 cells (a hippocampal cell line established from prnp-ablated mice [15]). We detected the NH 2 -terminal fragment predominantly in intracellular compartments, while the COOHterminal fragment was predominantly detected at the cell surface membranes, overlapping with lipid rafts. The full length PrP C showed a merged color of both terminals in the Golgi compartments. The NH 2 -terminal fragment of PrP C seems to reside inside vesicles, which may not reflect a distribution within any single specific organelle [13], where integral membrane and linker proteins would be required for interaction with MTs to bridge the luminal and cytoplasmic phases across membranes [16][17][18]. Accordingly we refer to this fragment hereafter as "vesicular GFP-PrP C ." Following transient transfection into N2a cells, vesicular GFP-PrP C exhibited an anterograde movement in the direction of the plasma membrane at a speed of kinesin super family KIF4 (140~ nm/ sec) and an inward retrograde movement at a speed of dynein (1,000~ nm/sec), as determined by real-time imaging studies. Kinesin and dynein inhibitors blocked the anterograde and retrograde movements of vesicular GFP-PrP C , respectively, and anti-kinesin antibody blocked its anterograde movement, whereas anti-dynein blocked its retrograde movement [19]. These data suggest a kinesin (KIF4)-driven anterograde and dynein-driven retrograde movement of vesicular GFP-PrP C ; in addition, residues 53-91 and 23-33 were indispensable for interactions with kinesin and dynein, respectively [13,19]. These results were obtained using undifferentiated cultured cells, but little is known about the behavior of intracellular PrP C in differentiated neuronal cells. We have recently established an N2a cell line that stably expresses GFP-PrP C and have further examined the intracellular trafficking of vesicular GFP-PrP C in these cells under the differentiated condition. These experiments have identified a significant reduction exclusively in the kinesin-driven anterograde, but not in the dyneindriven retrograde, velocity of vesicular GFP-PrP C trafficking in the outgrowing neurites.

Abstract
We previously demonstrated microtubule (MT)-associated kinesin-driven anterograde and dynein-driven retrograde trafficking of cellular prion protein in undifferentiated mouse neuro2a (N2a) cells. The NH 2 -terminal fragment of the fluorescent cellular prion protein residing inside vesicles (hereafter "vesicular GFP-PrP C ") exhibited an anterograde movement towards the direction of the plasma membrane at a speed of 140~ nm/sec, which is comparable to the velocity of KIF4-driven movement, and a retrograde movement inwardly at a speed 1,000 nm/ sec, which is comparable to the velocity of dynein-driven movement. We investigated the behavior of movement of vesicular GFP-PrP C in the neurite by first establishing N2a cells that stably expressed GFP-PrP C and treating these with NGF for neurite differentiation, followed by real-time imaging. In neurites, the anterograde kinesin-driven velocity of vesicular GFP-PrP C was selectively reduced to ~50 nm/sec, which is comparable to the velocity of KIF5, whereas retrograde dynein-driven velocity remained unchanged. Injection of anti-KIF5 antibody into differentiated N2a cells stably expressing GFP-PrP C inhibited the anterograde movement of vesicular PrP in neurites, which exhibited neuriteassociated bulges that lacked PrP C signals. These data suggest the involvement of a motor switch from KIF4 to KIF5 in PrP C movement in neurites.

Antibodies and drugs
Anti-PrP rabbit polyclonal antibody K3 was raised against the PrP peptides (amino acid residues 76-90 in mouse PrP). Anti-tubulin antibody DM1A were purchased from Sigma-Aldrich Japan KK, Tokyo, Japan. Anti-KIF5C antibody was purchased from Abcam plc, Cambridge, UK.

Cell cultures, DNA transfection, and drug treatments
GFP-PrP was constructed as previously described [13,19]. The resulting plasmid was designated as pcDNA3.1-GFP-PrP. N2a cells were obtained from the American Tissue Culture Collection and grown at 37°C in MEM medium supplemented with 10% fetal bovine serum. N2a cells were stably transfected with pcDNA3.1-GFP-PrP using a DNA transfection kit (Lipofectamine, Life Technologies, CA, USA). Following stable transfection, cells were selected and maintained with 1 mg/ml of G418 (Wako Pure Chemical Industries, Osaka, Japan). Before imaging analyses, cells were differentiated for 7 days in the presence of 200 ng/ml NGF (Sigma-Aldrich Japan KK, Tokyo, Japan).

Immunofluorescence microscopy
Indirect immunofluorescence analyses were performed on cells rinsed with PBS containing Ca 2+ and Mg 2+ [PBS (+)] and fixed with 10% formalin in 70% PBS (+) for 30 min at room temperature. After four washes with PBS (−), the fixed cells were incubated in 10% FBS in PBS (−) for 30 min at room temperature. Cells were then incubated for 1 h at room temperature with antibodies at desired concentrations. After four washes with PBS(−), the cells were incubated for 1 h at room temperature with secondary antibodies, which were diluted 1:200 in PBS. The stained cells were washed four times with PBS(−) and mounted with SlowFade Antifade (Life Technologies, CA, USA). Samples were imaged using the Delta Vision microscope system (Applied Precision, Inc., Issaquah, WA, USA). Out-of-focus light in the visualized images was removed by interactive deconvolution.

Live cell imaging
Cells were cultured on 3.5 cm glass-bottom dishes (Matsunami Glass Ind., Ltd., Tokyo, Japan) and imaged using the Delta Vision microscopy system (Applied Precision Inc., Issaquah, WA, USA) equipped with an Olympus IX70 camera (Olympus Imaging Corp., Tokyo, Japan). Fluorescence signals were visualized using a quad beam splitter (Chroma Technology Corp., Rockingham, VT, USA ).

Antibody transfection
Antibodies were transfected into N2a cells using Chariot (Active Motif, CA, USA) following the manufacturer's protocol. In brief, 2 × 10 5 N2a cells were cultured in 3.5 cm glass-bottom dishes. Chariot (6 μl) diluted in DMSO (94 μl) was combined with 1.25 μg antibodies resuspended in 100 μl of PBS(−) and incubated at room temperature for 30 min. The cells were washed twice with 2 ml of PBS(−), and the Chariot-antibody complex was mixed with 400 μl Opti-MEM I medium (Life Technologies, CA, USA). The cells were incubated at 37°C in 5% CO 2 for 2 h and imaged using the Delta Vision microscopy system.

Results and Discussion
A cell line that stably expressed GFP-PrP was established by transfecting N2a cells with the plasmid and culturing the transfected cells in the presence of G418 ( Figure 1A). The cells that stably expressed GFP-PrP exhibited the same expression pattern as the cells that showed transient expression ( Figure 1B). When we added NGF to the stable expression cell line, the neurites extended and vesicular GFP-PrP C was localized within the neurite. In contrast, undifferentiated cells contained vesicular GFP-PrP C predominantly in intracellular compartments, revealing a dot-like distribution pattern ( Figure 1C). Immunofluorescence microscopy observation of GFP-PrP C along with endogenous PrP C with MTs (by anti-PrP polyclonal antibody K3/antitubulin monoclonal antibody DM1A) revealed that PrP C was localized on MTs in differentiated (Figures 2E-2H) and undifferentiated-N2a cells (Figures 2A-2D). This immunostaining profile supports our previous proposal that vesicular PrP C consists of the NH 2 -terminal PrP C fragment and interacts with the MTs [13].
The intracellular trafficking of vesicular GFP-PrP C was then examined by real-time imaging using NGF-differentiated and stably GFP-PrP expressed N2a cells. As shown in Figure 3, the neurite of the differentiated cells showed a reduction in the velocity of anterograde movement of vesicular GFP-PrP C toward the plasma membranes (i.e., the speed was ~50 nm/sec, compared to the velocity of cell body movement of 140~ nm/sec). In contrast, the retrograde velocity in the neurite remained the same as that in the cell body.
The observed anterograde velocity of vesicular GFP-PrP C (50~ nm/s) was comparable to the speed of KIF5-driven movement in the neurites. Therefore, next we examined the possible involvement of motor protein KIF5 for the anterograde movement of vesicular GFP-PrP in the neurite by injecting KIF5 antibody into the cells. As shown in the (Figure 4 compartmentation that occurs distant from the cell body, such as in synapses and neurites. For example, PrP C is expressed at high levels in synapses, suggesting an important role in neuronal function that might have vital implications for synaptic homeostasis [20] including synaptic inhibition [21] and copper metabolism [22]. In the neurites, interactions between the neural cell adhesion molecule (NCAM) and PrP C promote neurite outgrowth [23]. The establishment of an N2a cell line that stably expressed GFP-PrP C allowed us to perform detailed real-time imaging analysis of vesicular GFP-PrP C ; in this case, PrP C trafficking in live N2a cell neurites. The most significant finding was that the velocity of anterograde kinesin (KIF4)-driven movement was reduced by half and fell within the range of KIF5-driven motility, while the retrograde dynein-driven movement remained unchanged.
Motor proteins, such as the MT-based kinesin superfamily proteins (KIFs), are differentiated in the neurites and the cell body, possibly in a cargo-specific manner [24]. Within the kinesin superfamily, KIF4 conveys cargos from the minus to the plus ends of the MTs, a direction that corresponds to anterograde movement, at a speed of <200 nm/sec [25]; this movement is concentrated in the cell body [26]. In contrast, KIF5 conveys cargos (for example containing AMPA-type glutamate receptors [27]) in the neurites at a speed of approximately 50 nm/sec [28]. The velocities of plus-end-directed, kinesin-driven movement are thought to vary for a number of reasons [29]. This variation might derive from the complicated environment, where local obstacles such as actin filaments that could impede cargo movement. In addition, the functioning of multiple motors (including dynein) on a given cargo that could load the single kinesin motors, or a reduction in the number of active motors, could lead to lowered cargo movement velocities [30,31].
On the other hand, an alternative view holds that changing the number of motors should not affect the velocity of a vesicle if the motors are kinesins. This view originates from early kinesin gliding assays conducted in buffer, which showed that the velocity is constant over a broad range of kinesin surface densities [32]. Thus, the mechanism by which transported vesicles change their speed remains controversial. Our observations suggest that the motor switch between KIF4 and KIF5 is an underlying mechanism for the velocity change that occurs in a subcellular localization-dependent manner. The precise switching mechanisms of KIF motors, i.e., whether the mode of PrP C trafficking observed in the current study is a widespread phenomenon, remains an intriguing question [25,33].