Dynamic behavior of GFP-GRASP55 in accumulated retrograde tubules.

<p>NRK cells transiently expressing GFP-GRASP55 were held in a microscope stage at 37°C, and were treated with 5 μg/ml BFA in conjunction with 100 μM myr-PKI-A (A; n = 10) or 30 μM H-89 (B; n = 15). Live cells were examined by fluorescence microscopy, and the time after initiation of each treatment is shown in the upper right corner of each panel in minutes:seconds. In A, arrowheads indicate localization of GFP-GRASP55 at ER exit sites that during the treatment seems to vanish; large arrows indicate the elongation of a tubule (21:00–21:30) that retracts and dilates at a central portion (37:10), and the dilated part remains steady until the end of imaging (51:00); and small arrows indicate a small tubule that elongates at a later time (37:10). In B, small arrows indicate elongation of tubules at different times (19:50, 22:00, 22:50, 26:10, and 33:30), the curving and retraction of a tubule (23:30 to 24:50), the elongation and retraction of a tubule from the central portion of an accumulated tubule (26:10 to 28:10), and the elongation, curving, and retraction of another tubule (33:30 to 34:20); and arrowheads indicate the dilated tip of an accumulated tubule. Bars, 5 μm.</p

Protein Kinase A Activity Is Necessary for Fission and Fusion of Golgi to Endoplasmic Reticulum Retrograde Tubules

<div><p>It is becoming increasingly accepted that together with vesicles, tubules play a major role in the transfer of cargo between different cellular compartments. In contrast to our understanding of the molecular mechanisms of vesicular transport, little is known about tubular transport. How signal transduction molecules regulate these two modes of membrane transport processes is also poorly understood. In this study we investigated whether protein kinase A (PKA) activity regulates the retrograde, tubular transport of Golgi matrix proteins from the Golgi to the endoplasmic reticulum (ER). We found that Golgi-to-ER retrograde transport of the Golgi matrix proteins giantin, GM130, GRASP55, GRASP65, and p115 was impaired in the presence of PKA inhibitors. In addition, we unexpectedly found accumulation of tubules containing both Golgi matrix proteins and resident Golgi transmembrane proteins. These tubules were still attached to the Golgi and were highly dynamic. Our data suggest that both fission and fusion of retrograde tubules are mechanisms regulated by PKA activity.</p></div

Dynamic behavior of GalT-YFP in accumulated retrograde tubules.

<p>NRK cells transiently expressing GalT-YFP were held in a microscope stage at 37°C, and were treated with 5 μg/ml BFA in conjunction with 100 μM myr-PKI-A (A; n = 10) or 30 μM H-89 (B; n = 15). Live cells were examined by fluorescence microscopy, and the time after initiation of each treatment is shown in the upper right corner of each panel in minutes:seconds. In A, large arrows indicate the dwindling of an initial thick tubule that leaves a swollen central portion; and small arrows indicate the formation of a dilated tip. In B, arrowheads indicate the dilated tip of an accumulated tubule; large arrows indicate a tubule that elongates and retracts; and small arrows indicate the elongation and retraction of another tubule. Bars, 5 μm.</p

The tip of an accumulated tubule containing GM130 colocalizes with an ER exit site.

<p>NRK cells were left untreated (A) or treated with 5 μg/ml BFA (B), or with BFA in conjunction with 100 μM myr-PKI-A (C), for the time indicated. Cells were fixed, permeabilized, immunolabeled with mouse monoclonal antibody to GM130 and rabbit antibody to Sec23A, followed by Alexa-594-conjugated donkey anti-mouse IgG (red channel), and Alexa-488-conjugated donkey anti-rabbit IgG (green channel). Nuclei were stained with DAPI (blue channel). Stained cells were examined by confocal fluorescence microscopy. Merging of the images in the red, green, and blue channels generated the third image on each row; yellow indicates overlapping localization of the green and red channels. In B, arrows indicate colocalization at ER exit sites. In C, the arrow indicates colocalization of the tip of a tubule containing GM130 with an ER exit site. Bar, 10 μm.</p

P115 and GM130 localizes distinctly in accumulated retrograde tubules.

<p>NRK cells were left untreated (A) or treated with 5 μg/ml BFA (B), or with BFA in conjunction with 100 μM myr-PKI-A (C), for the time indicated. Cells were fixed, permeabilized, immunolabeled with rabbit polyclonal antibody to p115 and mouse monoclonal antibody to GM130, followed by Alexa-594-conjugated donkey anti-rabbit IgG (red channel), and Alexa-488-conjugated donkey anti-mouse IgG (green channel). Nuclei were stained with DAPI (blue channel). Stained cells were examined by fluorescence microscopy. Merging of the images in the red, green, and blue channels generated the third image on each row; yellow indicates overlapping localization of the green and red channels. In C, arrows indicate colocalization in accumulated tubules. Bar, 10 μm.</p

PKA activity in NRK cells is less sensitive to H-89 than in HeLa cells.

<p>NRK cells (A) or HeLa cells (B) were left untreated or treated with H-89 at the indicated concentrations for 60 min. Cell extracts were prepared to assess PKA activity using a non-radioactive protein kinase assay kit (Calbiochem), according to the manufacturer's instructions. PKA activity at each concentration of H-89 represents the percentage compared to the activity in untreated cells. N = 3.</p

Retrograde tubules accumulate upon PKA inhibition.

<p>NRK cells were left untreated (A) or treated with 5 μg/ml BFA (B-C), or with BFA in conjunction with either 30 μM H-89 (D) or 100 μM myr-PKI-A (E), for the time indicated. Cells were fixed, permeabilized, immunolabeled with mouse monoclonal antibody to GM130 and rabbit antibody to α-mannosidase II (Man-II), followed by Alexa-594-conjugated donkey anti-mouse IgG (red channel), and Alexa-488-conjugated donkey anti-rabbit IgG (green channel). Nuclei were stained with DAPI (blue channel). Stained cells were examined by confocal fluorescence microscopy. Merging of the images in the red, green, and blue channels generated the third image on each row; yellow indicates overlapping localization of the green and red channels. In B, arrowheads indicate the emergence of tubules containing GM130, but devoid of Man-II. In C, arrows indicate colocalization at ER exit sites. In D and E, arrowheads indicate colocalization at accumulated tubules, and arrows indicate colocalization at swollen portions of tubules. Bar, 10 μm.</p

PKA activity is necessary for the fission of accumulated tubules.

<p>(A) NRK cells were treated with 5 μg/ml BFA in conjunction with 100 μM myr-PKI-A (m-PKI-A). After 60 min, myr-PKI-A was washed out in the presence of BFA, and cells were fixed at the times indicated. Cells were permeabilized and immunolabeled with rabbit polyclonal antibody to GRASP65, followed by Alexa-488-conjugated donkey anti-rabbit IgG. Stained cells were examined by fluorescence microscopy. Large arrows indicate large cut tubules. Small arrows indicate small cut tubules. Bar, 10 μm. (B) NRK cells transiently expressing GFP-GRASP55 were held in a microscope stage at 37°C. Cells were treated with 5 μg/ml BFA in conjunction with 30 μM H-89, and H-89 was washed out in the presence of BFA (n = 7). The large arrow indicates the site where an accumulated tubule is cut; small arrows indicate a punctum where the cut tubule collapses. Bar, 5 μm. (C) NRK cells transiently co-expressing CFP-GM130 and Sec13-YFP were held in a microscope stage at 37°C. Cells were treated, and myr-PKI-A was washed out as in A (n = 5). Arrows in the upper panels indicate the collapsing of a cut tubule into an ER exit site indicated by a large arrow. Bar, 5 μm. In B and C, live cells were examined by fluorescence microscopy, and the time after initiation of the treatment is shown in the upper or lower right corner of each panel, respectively, in minutes:seconds.</p

Co-assembly of Viral Envelope Glycoproteins Regulates Their Polarized Sorting in Neurons

<div><p>Newly synthesized envelope glycoproteins of neuroinvasive viruses can be sorted in a polarized manner to the somatodendritic and/or axonal domains of neurons. Although critical for transneuronal spread of viruses, the molecular determinants and interregulation of this process are largely unknown. We studied the polarized sorting of the attachment (NiV-G) and fusion (NiV-F) glycoproteins of Nipah virus (NiV), a paramyxovirus that causes fatal human encephalitis, in rat hippocampal neurons. When expressed individually, NiV-G exhibited a non-polarized distribution, whereas NiV-F was specifically sorted to the somatodendritic domain. Polarized sorting of NiV-F was dependent on interaction of tyrosine-based signals in its cytosolic tail with the clathrin adaptor complex AP-1. Co-expression of NiV-G with NiV-F abolished somatodendritic sorting of NiV-F due to incorporation of NiV-G•NiV-F complexes into axonal transport carriers. We propose that faster biosynthetic transport of unassembled NiV-F allows for its proteolytic activation in the somatodendritic domain prior to association with NiV-G and axonal delivery of NiV-G•NiV-F complexes. Our study reveals how interactions of viral glycoproteins with the host's transport machinery and between themselves regulate their polarized sorting in neurons.</p></div

Live-cell imaging shows that NiV-G increases axonal transport of NiV-F.

<p>(A–C) Single-frame images from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107.s006" target="_blank">Video S1</a> (thin upper images) and kymographs (bottom images) of the analysis of particles moving along 100 µm of axons in DIV10 rat hippocampal neurons co-transfected on DIV5 with plasmids encoding either NiV-F-GFP (NiV-F) and mCh-Tub (Tub) (panel A), NiV-G-mCh (NiV-G) and GFP (panel B) or NiV-F-GFP and NiV-G-mCh (panel C). Images in (A) and (B) are shown in grayscale. In panel (C), NiV-F-GFP and NiV-G-mCh fluorescence are shown individually in grayscale and as green and red, respectively, in merged images (yellow indicates co-localization). Tracings with negative and positive slopes in kymographs represent anterograde and retrograde movement of particles, respectively; vertical lines represent particles that are stationary during the 60 s recording. (D) Quantification of NiV-F-GFP and NiV-G-mCh axonal transport in neurons expressing these proteins individually or in combination. Data shown represent the number of anterograde (Ant), stationary (Stat) and retrograde (Ret) particles per 100 µm of axon length during the 60 s recording. NiV-F and NiV-G (green and red bars, respectively) show the number of axonal particles containing these two proteins when expressed individually. “NiV-F (+NiV-G)” (light green bars) is the number of NiV-F-GFP particles in neurons co-expressing NiV-G-mCh; “NiV-G (+NiV-F)” (salmon bars) represents the number of NiV-G-mCh particles in neurons co-expressing NiV-F-GFP. Values are the means±SEM of 20–22 independent measurements for each condition and represent the total number of particles (n_p) indicated under the graph. Statistical significance was calculated by one-way ANOVA followed by Dunnett's test. (*) <i>P</i><0.01 when compared to NiV-F-GFP-containing particles in cells expressing only this protein. (E) Single-frame images from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107.s007" target="_blank">Video S2</a> showing dendritic particles from neurons co-expressing either NiV-F-GFP and mCh-Tub, NiV-G-mCh and GFP, or NiV-F-GFP and NiV-G-mCh (upper panels in grayscale; bottom panel with merged NiV-F-GFP and NiV-G-mCh images in green and red, respectively). The lower color image is a 4× magnification of the boxed area in the above image; color arrows point to particles containing either NiV-F-GFP or NiV-G-mCh (green and red, respectively) or both proteins (yellow). Scale bar: 10 µm. (F) Co-localization of NiV-F-GFP and NiV-G-mCh in axonal and dendritic particles of co-transfected neurons. NiV-F-GFP co-localization in co-transfected neurons (NiV-F (+NiV-G), light green bars) was the percentage of NiV-F-GFP-containing particles that also contained NiV-G-mCh. Similarly, NiV-G-mCh co-localization (NiV-G (+NiV-F), salmon bars) was the percentage of NiV-G-mCh-containing particles that also displayed NiV-F-GFP. Values are the means±SEM of 22 and 33 measurements of axonal and dendritic particles, respectively, and represent the total number of axonal and dendritic particles (n_pA and n_pD) containing NiV-F-GFP and NiV-G-mCherry indicated under the graph.</p