How many rough endoplasmic reticulum

Transcription is a nuclear event wherein mRNA template, encoding the sequence of the protein in the form of a trinucleotide code, is transcribed from DNA to provide a template for translation. Translation is a cytoplasmic process and the site of translation is the ribosomes. There, the amino acids are added by tRNAs and then are linked together in a specific order as specified in the mRNA transcript.

Subsequent to these events are maturation processes, such as proteolysis , post-translational modification , and protein folding. In the early phase of translation, a signal peptide is synthesized i. The signal is an indication that the protein is for further processing in the ER. When this signal is recognized by a signal recognition particle the ribosome translating the protein docks to the endoplasmic reticulum via the translocon.

The ribosome, then, returns back to the translation of the protein. The chain continues to grow as the mRNA transcript is translated through the docked ribosome. The chain eventually makes its way into the ER through the translocon that spans across the ER membranes.

The signal peptide is removed by a signal peptidase in the lumen of the ER. The nascent protein is folded in the ER by the chaperone proteins e. The properly-folded protein is then packed into a transport vesicle to be shuttled to the Golgi apparatus where it would undergo maturation for transport along the cytoskeleton to other cytoplasmic organelles like lysosomes and peroxisomes or for secretion out of the cell.

Some of the proteins synthesized inside the ER will be retained, such as those that become part of the ER membrane. Those that are retained in the ER have a retention motif, e. An unfolded or misfolded protein triggers an endoplasmic reticulum stress response.

Vectors to fuse the FP to the C-terminus of proteins were also created. Again cloning sites and a flexible linker were added.

In this case the FP coding region was amplified with the stop codon intact. The vectors generated were pYC and pCC. To these basic vectors, promoters were added into the polylinker present from the starting vector. Each promoter was cloned into the four fusion vectors so that there were sets of vectors, for example, p g YC, p g YN, p g CC, p g CN, all of which contained a particular promoter, in this case glr To make plasmids that expressed FP-fusion proteins in worms, genomic coding regions, amplified from cosmids or genomic DNA, of predicted proteins were inserted into vectors like p g YC.

Complete coding regions were used except for Golgi markers. The sequence names for the predicted ER proteins are listed in Figure 3 D. The location of the tag is also indicated in Figure 3 D. For the NE marker, full-length emerin M01D7. Similarly the Golgi marker, mans for mannosidase-short was tagged at the C-terminus with the FP. In this case, however, only a short region of the coding sequence was PCR amplified. The fusion protein is predicted to contain 82 amino acids from the sequence F58H1.

For observation, C. A coverslip was placed on top, excess agarose was cut away, and the coverslip was sealed with nail polish. Worms were observed between 10 and 60 min after mounting. Generally L2 or L3 worms were analyzed. Frame interlace collection was used for most images, except Figure 2 B for which line interlace collection was used. Images of the whole cell were collected before the bleach, immediately after, and every 10 s thereafter.

Quantitation was performed using NIH Image 1. Total pixel intensity was summed in a background region of the image, part of the bleached region of the cell, and part of the unbleached region for each time point. Background was subtracted from both bleached and unbleached values, and then the ratio of bleached to unbleached was taken for each time point and divided by the initial prebleach ratio to correct for difference in intensity between regions of the cell.

A computer program that analyzes diffusion in complex structures Siggia et al. General TEM methods have been described previously Hall, To accentuate the staining of ribosomes within neuronal cytoplasm, several fast freezing methods were explored, followed by freeze substitution and plastic embedment cf.

McDonald, ; Williams-Masson et al. Briefly, we used either metal mirror fixation or high-pressure freezing to quickly immobilize live animals on a piece of filter paper, inside a sealed piece of flexible dialysis tubing, or in a slurry of yeast or Escherichia coli. The frozen samples were then slowly exposed to a primary fixative of osmium tetroxide in methanol or acetone and, while still kept very cold, dehydrated through solvents, and infiltrated with plastic resin.

After curing, the animals were thin-sectioned and poststained for TEM by conventional means. We wanted to establish a system in which to study ER protein localization in multiple differentiated cell types.

To visualize the ER in different C. These fusions were expressed under the control of cell type—specific promoters. In various cell types, for example, body wall muscle Figure 1 A , the FP-ER fusion was localized to a reticular intracellular network that appeared similar to the ER in many other types of cells. In head muscle cells the distributions of predicted ER markers were compared with those of FP-fusions to proteins predicted to be targeted to other intracellular organelles.

FP-fusions to the stalk and transmembrane regions of two predicted Golgi resident enzymes were targeted to spots scattered throughout the cell Figure 1 B and our unpublished results , a pattern consistent with localization of Golgi proteins in other invertebrates for example, Drosophila [ Stanley et al. Thus FP-fusions to worm proteins can be constructed based on analogy with mammalian homologues, correctly targeted, and visualized in live cells.

A single confocal plane is shown, and the NE and surrounding reticulum are visible. B In head muscle markers for different cellular membranes are easily distinguishable. In the Ymans and YSP12 panels the nucleus is at the right, and the anterior contractile region of the cell is on the left. Predicted RER membrane proteins were chosen based on sequence similarity to mammalian proteins involved in translocation across the ER membrane.

General ER proteins were considered to be all those ER proteins involved in functions other than translocation across the membrane, for example, lipid synthesis. FP fusions to predicted RER and general ER proteins were expressed in hypodermal cells using the dpy-7 promoter, and in intestinal cells using the general promoter rpl In intestinal cells we occasionally saw patches of membranes enriched only in PIS but they were most obvious in deteriorating worms.

Distribution of ER membrane proteins, and the ER itself, in hypodermal and intestinal cells. Several regions of the cell appear wavy because of movement of the worm during imaging. C A transverse section of a worm was examined by electron microscopy after immersion fixation.

A portion of a hypodermal cell is shown. The cytoplasm is filled with RER and free ribosomes. M, mitochondria; P, an infolding of the plasma membrane. The hypodermal cell is bounded on the right by cuticle. D A portion of an intestinal cell from a transverse section of a worm viewed by electron microscopy and fixed as in C is shown.

Ultrastructural analysis of C. We did not see any evidence of SER, and if it is present in these cells, it must account for only a small portion of the total ER. Because neurons in other organisms contain SER, neurons were chosen as a candidate cell type in which RER membrane proteins might be spatially segregated.

Neuronal membranes have been best studied in mammals in highly polarized neurons with axons and dendrites. To visualize the ER in C. The glr-1 promoter drives expression in different classes of motorneurons and interneurons Hart et al.

Most of the cell bodies are located in the ganglia near the nerve ring, although a few are in the retrovesicular ganglion posterior to the nerve ring, and some are in the tail ganglia. In addition to neuronal expression, the vectors with the glr-1 promoter gave some expression in several head muscles see Figure 1 B. The localization of different ER membrane proteins in neurons is distinct. Muscle cells in the nose of the worm M, cells at the tip of the nose on either side as well as some other head cells express the FP.

Fluorescence is also seen in neurons in several head ganglia H near the nerve ring, in the retrovesicular ganglion R , in tail ganglia T , and in neurites that project along the ventral nerve cord V. Neurites sweeping across the nerve ring are marked N. Neurites in the ventral nerve cord are labeled V. FP fusions were to the full-length genomic region of the gene listed.

The FP is represented by the barrel structure. The predicted topologies are shown with the lumen at the top of the diagram. To determine whether any difference in localization of RER and general ER membrane proteins could be detected in neurons, FP-fusions to the two classes of predicted proteins were expressed under the glr-1 promoter. The predicted general ER proteins were present in neurites as well as the cell body, whereas most of the predicted RER membrane proteins were concentrated in the cell body.

Representative confocal images of nerve rings from these two groups are shown in Figure 3 B. Slight fluorescence in the neurites is probably due to overexpression. More rigorous comparisons between different classes of ER membrane proteins were made in worms expressing two ER proteins in the same cells. With the microscopy setup used, cross-talk between the two channels is negligible White et al.

Because fusions to GFP variants can sometimes cause mistargeting of proteins and because the ER has not been studied in worms, a number of different FP-ER fusions were tested. The correlation between the predicted category of the protein and observed localization Figure 3 D strengthened the validity of the fusions as markers for different classes of ER proteins.

Of three predicted general ER proteins tested, all localized to both the cell body and neurites, consistent with the hypothesis that they incorporate into, and distribute throughout, the ER membrane. Of the five homologues of translocation proteins tested, four were enriched in the cell body. The FP-fusion to the kDa subunit of signal peptidase SP12 was distributed throughout the neurons like the general ER membrane proteins.

The signal peptidase complex has been reported to fractionate with rough membranes Vogel et al. Although the major difference in localization of ER membrane proteins in neurons was between those that were concentrated in the cell body and those that were not, several other differences were also observed. At present we have no explanation for these differences in individual proteins so we have focused on the broader distinction between general ER proteins and those involved in translocation.

Conclusions about localization of ER membrane proteins in neurons from these experiments require that the FP-tagged membrane proteins are stably localized to the ER. An alternate explanation for the difference in distribution between general ER markers and RER markers is that the general ER markers escaped to the plasma membrane. We consider this explanation unlikely.

Because the glr-1 promoter also drove expression in head muscle cells, all ER markers were examined in these cells as well. For each ER marker, a reticular pattern was observed in head muscle cells for example, see Figure 1 B. In these cells, and in the larger cells e. We tested the imaging conditions to make sure they were robust enough to reliably detect fluorescence in neurites and were not influenced by differences in the properties of YFP and CFP. Autofluorescence is always higher in the CFP channel; the blobs that are seen in many CFP images are autofluorescence and are unrelated to the fusion protein being expressed.

The second test we performed was to make reciprocally tagged pairs of fusion proteins and image both pairs. Therefore, the results obtained were independent of which protein was tagged with a particular GFP variant. Differences in the distribution of ER markers in neurons are independent of the imaging conditions used. B In all panels cells in the retrovesicular ganglion were imaged with the ventral nerve cord V passing close by.

Because most of the predicted RER membrane markers were concentrated in the cell body, we tested whether morphologically recognizable RER was also localized there. Electron micrographs of C. In contrast membranes studded with ribosomes are not seen in the neurites. In regions of synaptic contact many intracellular membranes are present within the neurite, most identifiable as synaptic vesicles. However, even in regions of the neurite that do not make synaptic contact, an intracellular membrane profile is often seen Figure 5 B.

This membrane profile is smooth ribosome-free and often visible in many consecutive sections. It varies in dimensions from section to section, shows irregular swellings along its length, and is always larger than the microtubules. The appearance of this membrane is consistent with it being SER.

At the ultrastructural level RER is observed in the cell body of C. A The cell body of a neuron in which the ER membranes have become slightly distended during fixation is shown. The distention of the ER makes it clear that the membranes are tightly covered with ribosomes, and highlights the connection of the peripheral ER with the NE.

Free ribosomes are also present. B A cross section of neurites in the ventral nerve cord is shown. Small regular circles are microtubules.

Larger irregular profiles are smooth membranes arrowheads. Scale bars, 0. Because RER membrane proteins often associate with ribosomes, we tested whether ribosomes were also concentrated in the cell body.

L23A was chosen to visualize ribosomes because a GFP-tagged version of the yeast homolog can rescue an L23A knockout in yeast Hurt et al. Localization to a region of the neuron was consistent with the tagged protein being incorporated into ribosomes.

The Golgi apparatus is also closely associated with the ER and recent observations suggest that parts of the two organelles, i. This is an extensive organelle composed of greatly convoluted but flattish sealed sacs, which are contiguous with the nuclear membrane.

These are called membrane bound ribosomes and are firmly attached to the outer cytosolic side of the ER About 13 million ribosomes are present on the RER in the average liver cell. Rough ER is found throughout the cell but the density is higher near the nucleus and the Golgi apparatus. This process is called translation. Certain cells of the pancreas and digestive tract produce a high volume of protein as enzymes.

Many of the proteins are produced in quantity in the cells of the pancreas and the digestive tract and function as digestive enzymes. Proteins are produced for the plasma membrane, Golgi apparatus, secretory vesicles, plant vacuoles, lysosomes, endosomes and the endoplasmic reticulum itself. Some of the proteins are delivered into the lumen or space inside the ER whilst others are processed within the ER membrane itself. In the lumen some proteins have sugar groups added to them to form glycoproteins.

Some have metal groups added to them. It is in the rough ER for example that four polypeptide chains are brought together to form haemoglobin. The rough endoplasmic reticulum has on it ribosomes, which are small, round organelles whose function it is to make those proteins. Sometimes, when those proteins are made improperly, the proteins stay within the endoplasmic reticulum. They're retained and the endoplasmic reticulum becomes engorged because it seems to be constipated, in a way, and the proteins don't get out where they're suppose to go.