Preadolescent presentation of a lumbar chordoma: results of vertebrectomy and fibula strut graft reconstruction at 8 years

Chordoma is a tumour of notochordal origin which usually involves the sacrum or skull base presenting in adulthood. Chordoma in a mobile spinal segment is infrequent and the authors report an extremely rare presentation of L3 chordoma in a child aged 7 years. Although a benign tumour, mobile segment chordoma is more locally aggressive, more likely to metastasise and has a poorer 5 year survival than sacral and clival lesions. Wide surgical excision and reconstruction is the treatment of choice in vertebral chordoma. This case was treated with staged vertebrectomy and fibular strut graft reconstruction and the results of clinical and radiological follow up at 8 years are presented.
Case report
A previously well 7-year old child presented with a 6 month history of right anterior thigh and knee pain. Examination revealed a marked list to the right, painful restriction of spine movements in all directions, an absent right knee jerk with reduced sensation in the right L3 distribution. There was no motor weakness or sphincter disturbance. Back pain restricted straight leg raise bilaterally to 30°. No history of weight loss, systemic symptoms and no past medical and family history of relevance. Blood tests were within normal limits. Inflammatory markers were not elevated. Plain radiographs revealed an osteolytic lesion in the posterior body of L3. MRI of the whole spine identified an isolated lesion at L3 with right pedicle involvement and tumour extension anterior to the dural sac at L4 (Fig. 1). Isotope bone scan showed no increased uptake. A staging CT of the chest showed no evidence of metastases.
Fig. 1Pre-operative MRI scan of lumbar spine showing the lesion involving L3 vertebra
Transpedicular biopsy at L3 was performed and histology revealed fragments of remodelling bone, haemorrhage and a myxoid tissue containing strips and ribbons of cohesive, vacuolated cells. These cells had the appearance of notochordal-type tissue. Immunohistochemistry was positive for Cytokeratin S-100.
In view of the histological findings and atypical young age of presentation a radical 360° L3 vertebrectomy with pedicle screw stabilization and fusion was planned.
At operation the L3 tumour had extended through the lamina and transverse process on the right compromising the right L3 nerve root. There was both cranial and caudal extradural extension of the tumour anterior to the dural sac with tenting from L2 to L4. A wide decompression was performed with resection of the posterior elements and partial posterior vertebrectomy at L3. To ensure that the tumour within the spinal canal was completely excised it was necessary to resect the inferior lamina of L2 and most of the lamina at L4 bilaterally. Hence it was felt appropriate to extend the instrumentation inferiorly to L5.
Pedicle screw instrumentation was performed at L2 and L5 bilaterally. Free fat graft was placed over the dura and nerve roots. Bone graft was placed posterolaterally from L2 to L5 (Fig. 2). An epidural infusion was used postoperatively for pain relief. Initial post operative neurological assessment was normal.
Fig. 2AP and lateral X-rays following first stage of the procedure
The following day deterioration in her lower limb neurological status was noted with no active movement or sensation in her lower limbs. That this was not recognised immediately was in part attributed to her epidural and to some difficulty with neurological assessment in a young child with systemic analgesia after major surgery. She was returned to theatre where a large haematoma was found and drained. Although subsequent sensory recovery was good, her motor recovery was slow with grade 1 power in her lower limbs.
Two weeks later, once progressive neurological recovery was established, she underwent second stage anterior vertebrectomy of L3 through a retroperitoneal approach. Unilateral L2, L3 and L4 segmental vessels were ligated to expose L3. A complete excision of the remaining L3 and posterior longitudinal ligament was performed in order to adequately decompress the dural sac anteriorly. Three fibular strut autografts from L2 to L4 were used to reconstruct the anterior column. Posterior iliac crest graft was used for further augmentation (Fig. 3).
Fig. 3AP and lateral X-rays following second stage of the procedure showing the fibular strut grafts used
Neurological recovery continued post-operatively and she had regained normal sensation and grade 5 motor power in both lower limbs by 6 months. Bladder sphincter dysfunction has persisted, requiring intermittent self catheterisation and anti-cholinergic medication. She was nocturnally continent. Bowel function remained normal throughout.
Radiographic fusion was evident at 6 months. Metal work was removed at 12 months and intra-operative biopsy showed no tumour recurrence (Fig. 4).
Fig. 4AP and lateral X-rays following removal of metal work showing consolidation of the strut grafts
She remained under annual follow-up with MRI and plain radiographs. At 8 years she has no evidence of tumour recurrence. Her neurological status is unchanged. Imaging has shown excellent remodelling of the graft (Figs. 5, 6). She is skeletally matured with no lower limb malalignment and experienced no fibular donor graft site morbidity.
Fig. 5Chronological X-rays showing graft incorporationFig. 6Eight year follow-up MRI Scan
Discussion
Chordoma is a primary bone tumour arising from notochord remnants. It is a rare tumour accounting for only 1–4% of primary bone tumours [1]. Chordoma is usually a slow growing tumour, often with local recurrence and it may metastasise late in its course [1]. Typically it presents in late middle age and may occur anywhere along the spinal column. Chordoma is found most commonly in the clivus, sacrum and infrequently in the mobile vertebrae [2].
The optimum treatment for vertebral chordoma is wide surgical excision. Radiotherapy may provide short term benefit when adequate excision is not possible and in cases of local recurrence [3]. Surgical treatment has become more aggressive in recent years evolving from intralesional debulking to en bloc resection, as documented by Boriani et al., over a 45-year period in their treatment of 21 cases of chordoma located in the spine above the sacrum [4].
Chordomas are considered to be low grade tumours and metastases are infrequent at presentation [1, 5]. The prognostic value of staging is therefore diminished, compared to the value of staging in other bone tumours, because all these tumours are stage IA or IB according to the Musculoskeletal Tumor Society Staging System [6].
Mobile segment chordomas have a 50% survival rate at 5 years and a 28% survival rate at 10 years [4, 7]. Sacral chordomas on the other hand are reported as having 86% 5 year survival [3]. Treatment outcome is significantly influenced by the size and site of chordoma [1, 4]. Chordomas found in the vertebral bodies appear to be more aggressive than those arising in the clivus or the sacrum [3]. Metastases have been reported in 80% of the vertebral body chordomas, as compared to a rate of 43% of all chordomas [3]. However, the survival rate appears to be affected more by local tumour progression than by metastases [1].
Conclusion
This case is unusual given the young age at presentation. She is the youngest patient reported in world literature. Despite the poorer published results of mobile segment chordomas, radical surgical excision and reconstruction has resulted in an 8 year disease free survival. Fibular strut graft reconstruction proved an excellent reconstructive option in the growing skeleton with potential for remodelling. It is likely that the young age has contributed to the near complete neurological recovery.

chordoma
vertebrectomy
mobile spinal segment
benign tumour
fibular strut graft reconstruction


Prior outpatient antibiotic use as predictor for microbial aetiology of community-acquired pneumonia: hospital-based study

Objective The causative micro-organism in community-acquired pneumonia (CAP) is often difficult to predict. Different studies have examined chronic morbidity and clinical symptoms as predictors for microbial aetiology of pneumonia. The aim of our study was to assess whether prior outpatient antimicrobial treatment is predictive for determining the microbial aetiology of CAP.
Introduction
Community-acquired pneumonia (CAP) remains a major reason for hospital admission and a common cause of death in developed countries [1, 2]. The initial management of patients hospitalised with pneumonia consists mostly of empirical antimicrobial treatment [3, 4]. Determination of the appropriate antimicrobial treatment is essential as inadequate antimicrobial treatment, generally defined as microbial ineffective therapy against the causative pathogen, can negatively influence patient outcome [5]. Because it is not always possible to identify the causative pathogen, especially during the first days following hospitalisation, many studies have focussed on other parameters suggestive of the causative pathogen. The most frequently studied parameters for this purpose are patient characteristics (age and co-morbidities) and clinical signs [6, 7], although nonresponsiveness to prior outpatient antimicrobial treatment has also been suggested as a predictor of the aetiology of pneumonia. However, to date, this latter parameter has not been comprehensively quantified and documented. The aim of the study reported here was to assess whether prior outpatient antimicrobial treatment is a predictor of microbial aetiology in patients admitted to hospital for CAP.
Methods
The study was conducted in the St. Antonius Hospital, a 600-bed teaching hospital in Nieuwegein, The Netherlands.
Patient population
This was a prospective observational study of patients with confirmed pneumonia admitted between October 1, 2004 and August 1, 2006. Pneumonia was defined as a new or progressive infiltrate on a chest X-ray plus at least two of the following criteria: cough, sputum production, temperature >38°C or <35°C, ausculatory findings consistent with pneumonia, leucocytosis or leucopenia (>10 g/l, <4 g/l or >10% rods in leucocyte differentiation), C-reactive protein >3 times the upper reference value for normal. Patients who were immune compromised (systemic steroid use at admission (prednisone equivalent >20 mg/daily for more than 3 days), haematological malignancies and other immunosuppressive therapy) were excluded. The study was approved by the local Medical Ethics Committee, and informed consent was obtained from each patient.
Microbial aetiology workup
At least two blood cultures were performed, and sputum was taken for Gram-staining and culture and subsequently analysed by Taqman real-time PCR for Mycoplasma pneumoniae, Legionella pneumophila and Chlamydophyla psittaci [8]. Pharyngeal samples were taken for viral culture. Urine was sampled for antigen testing on Streptococcus pneumoniae and L.pneumophila (Binax NOW; Binax, Scarborough, ME) [9, 10]. In addition, serum samples taken on the day of admission and on day 10 were analysed in pairs for detection of a fourfold rise of antibodies to respiratory viruses, Coxiella burnetii, M. pneumoniae, and C. psittaci by complement fixation assay [11]. For each patient, the total workup was completed, and the microbiology department was blinded to data on outpatient antibacterial drug use. When both viruses and bacteria were identified in a patient, the search prevailed for a definite aetiology for the bacteria.
Exposure to antimicrobial therapy
Data on outpatient antimicrobial drug use were acquired through community pharmacy dispensing records that captured all drug exposures the year prior to hospital admission. A patient was considered to be exposed to an antimicrobial drug when a prescription was filled within 14 days prior to hospitalisation. The name, dosage and amount of antimicrobial drug dispensed were also ascertained. The prescribed antimicrobial drug was classified as appropriate or inappropriate in accordance with current Dutch guidelines on the initial treatment of patients with suspected pneumonia [12, 13].
Co-morbidity assessment
In addition to outpatient antimicrobial drug use, co-morbidities and other relevant patient characteristics were identified to address factors related with the aetiology of CAP. Co-morbidities were defined based on the presence of conditions for which the patient was under active medical supervision or was receiving treatment at the time of hospital admission. The co-morbidities evaluated were pulmonary diseases (chronic obstructive pulmonary disease or treated asthma), congestive heart failure, diabetes (both type I and type II), history of stroke and end-stage renal disease (serum creatinine >150 μmol/l). The patients were also classified according to the Pneumonia Severity Index (PSI) developed by Fine et al. [14]. This index classifies patients into five categories of predicted mortality risk (with the fifth category being that of highest mortality risk). The outpatient use of oral corticosteroids and gastric acid-suppressing drugs was also ascertained.
Statistical analysis
The SPSS statistical package (ver. 12.0.1 for Windows; SPSS, Chicago, IL) was used for the statistical analyses. Continuous data were expressed as the mean ± SD or as the median (interquartile range) where appropriate. To study the association between prior outpatient antimicrobial treatment and the aetiology of pneumonia, we applied multivariate logistic regression analyses. The analyses were conducted for overall aetiology and relevant pathogens separately. All baseline characteristics were considered potential predictors. Potential predictors were included in the multivariate model when they were retained after backward stepwise elimination. Significance was set at a p value < 0.05. The model’s performance (goodness-of-fit and discriminative ability) was tested by performing the Hosmer and Lemeshow test and calculating the area under the receiver operator characteristic (ROC) curve.
Results
In total, 201 patients with pneumonia were included in the study. The mean age of the patients was 63 ±17 years, and 124 patients were male. Three patients (1%) were admitted from a nursing home. Pneumonia severity as well as co-morbid illnesses are summarised in Table 1. The overall median duration of the hospital stay was 10 days [7 –14], and 21 patients were admitted to the intensive care ward. During the hospital stay, ten patients died, all due to pneumonia. The overall 28-day mortality rate was 5%. Forty-seven patients (23%) had received antimicrobial treatment in the 14-day time-window prior to hospital admission. The antimicrobial drugs dispensed to these patients are summarised in Table 2.
Table 1Demographics, co-morbidities and pneumonia severity index of 201 patients with community-acquired pneumonia (CAP)Characteristicn (%)Age (Years)  <6074 (37)  60–6939 (19)  70–7950 (25)  >8038 (19)Gender  Male124 (62)  Female77 (38)Co-morbidities  Pulmonary diseases71 (35)  Heart failure18 (9)  Diabetes35 (17)  History of stroke17 (9)  End-stage renal disease10 (5)  Nursing home resident3 (1)Co-medication  Oral corticosteroids58 (29)  Gastric acid suppressing drugs61 (30)Fine score at admissiona  I30 (15)  II34 (17)  III53 (26)  IV56 (28)  V28 (14)aFine et al. [14]Table 2Outpatient antibiotics utilization profile prior to hospitalisation for CAPType of antimicrobial drugNumber of users (%)aAppropriatebClavulanic acid18 (38)YesAmoxycillin12 (26)YesDoxycycline7 (15)YesClarithromycin5 (11)YesCo-trimoxazole4 (8)NoCiprofloxacin2 (4)NoNorfloxacin1 (2)NoAzithromycin1 (2)YesaTotal percentage exceeds 100% because some patients (n = 3) had two prescriptionsbBased on current Dutch guidelines NVALT and SWAB [12, 13]
The majority of the patients (79%) had their prescription filled within 4 days prior to hospital admission, and 85% of the prescribed antimicrobial drugs complied with current Dutch guidelines [12, 13]. A microbial aetiology could be determined in 128 (64%) of the patients. Table 3 shows the results of different tests used to determine the aetiological diagnosis of CAP.
Table 3Results of of different tests used to determine the aetiology of CAP Sputum cultureSputum PCRAntigen testingBlood cultureSerologyViral cultureNumber of samples1487818318213088Number of positive samples781436193814Percentage of positive samples531820102916Streptococcus pneumoniae33–3017––Gram-negative strain  Heamophilus influenzae19––0––  Other10––1––Atypical  Mycoplasma pneumoniae–7––8–  Legionella spp.156–7–  Other–2––1–Viral––––2214Other  S. aureus6––1––  Gram-positive other2––0––
Fewer causative pathogens were found in the population hospitalised following prior outpatient antimicrobial treatment than in patients without prior antimicrobial treatment [57 vs. 66%; crude odds ratio (OR) 0.71, 95% confidence interval (CI) 0.36–1.38]. In patients with prior beta-lactam treatment, aetiology in the group comprising atypical bacterial pathogens was more probable (eight of 29 cases, 28%; crude OR 4.66, 95% CI 1.73–12.56). The aetiology of S. pneumoniae was less prevalent in patients with prior beta-lactam antimicrobial treatment (four of 29 cases, 14%; crude OR 0.33, 95% CI 0.11–0.99). In multivariate analyses, these associations remained significant (OR 3.51, 95% CI 1.25–9.99 and OR 0.30, 95% CI 0.10–0.90, respectively) (Table 4). Additional significant predictors included in these models were heart failure, pulmonary comorbidity, pneumonia severity index and the use of gastric acid suppressing drugs. The goodness-of-fit of both multivariate models was good, with a p value of 0.899 (Hosmer and Lemeshow test) for the model predicting pneumococcal pneumonia and a p value of 0.995 for the model predicting pneumonia of an atypical aetiology. The corresponding areas under the ROC curve were 0.62 and 0.67, respectively.
Table 4Odds ratios (OR) for aetiology and prior outpatient beta-lactam treatment in patients admitted to hospital for CAPAetiologyPrior outpatient beta-lactam treatment (n = no. of patients)OR (95% CI)Yes (%)No (%)Total no. of samples29 (100)172 (100)Univariate  Pneumococcal4 (14)56 (33)0.33 (0.10–0.99)  Atypical8 (28)13 (8)4.66 (1.72–12.56)  Viral2 (7)14 (8)0.84 (0.18–3.89)  Gram-negative strains2 (7)21 (12)0.53 (0.12–2.40)  Other1 (3)7 (4)0.84 (0.10–7.11)  Unidentified12 (41)61 (36)1.28 (0.58–2.87)Multivariate  Pneumococcal––0.30 (0.10–0.90)a  Atypical––3.51 (1.25–9.99)bOR, Odds ratio; CI, confidence IntervalaAdjusted for heart failure and use of gastric acid suppressing drugsbAdjusted for pulmonary diseases and pneumonia severity index
In patients aged <60 years without co-morbidities, the aetiology of atypical bacterial pathogens was more prevalent (OR 4.64, 95% CI 1.72–12.56). Pulmonary co-morbidity was associated with the finding of S. pneumoniae and H. influenzae as causative pathogens (OR 1.87, 95% CI 1.00–3.47; OR 3.72, 95% CI 1.20–11.57, respectively). Patients who received macrolides prior to hospitalisation had an increased probability of viral pneumonia (two of five cases, 40%) (crude OR 8.67, 95% CI 1.34–56.23).
Discussion
The results of our study show that among patients admitted for CAP, a threefold decreased chance of having a S. pneumoniae infection and a threefold increased probability of having pneumonia of atypical aetiology were associated with the patient having received an initial beta-lactam treatment as an outpatient. These findings indicate that information on prior outpatient antimicrobial therapy has a predictive value in the diagnostic workup aimed at identifying the causative pathogen and planning the corresponding treatment in patients with pneumonia.
The initial management of patients hospitalised with pneumonia has been under constant study in different settings during the past decades. The choice of antimicrobial treatment, time to first antimicrobial drug administration and route of administration have all appeared to be relevant factors associated with the outcome of pneumonia [15–17]. A knowledge of the predominant microbial patterns in CAP is therefore essential when choosing an essential empirical antimicrobial treatment. Previous studies have found that S. pneumoniae, H. influenzae, Influenza virus A and B, Legionella spp. and C. pneumoniae are the most frequent pathogens in CAP [13], which is in accordance with our results. Because S. pneumoniae is the most frequently appearing pathogen, the administration of beta-lactam antibiotics is the initial empirical antimicrobial treatment of choice in the treatment guidelines on CAP [3, 12, 13]. Beta-lactam antibiotics, however, do not cover Legionella spp., C. pneumoniae and M. pneumoniae, the so-called atypical pathogens. Therefore, patients with pneumonia of atypical aetiology who are treated with beta-lactam antibiotics as an outpatient will probably not respond to treatment, with the possible consequence being a deterioration of the situation and subsequent hospital admission. Our finding of an increased prevalence of atypical pathogens in patients with prior outpatient beta-lactam treatment supports such an explanation, but also confirms what has already been suggested in the different guidelines for the management of community-acquired pneumonia in adults [13, 18]. These guidelines state that following the failure of the initial empirical treatment with beta-lactam antibiotics, the microbiological examination should be reassessed with a view to excluding the less common pathogens, such as atypical pathogens, and that antimicrobial treatment covering atypical pathogens should be considered. Our study supports the choice of antimicrobial treatment covering atypical pathogens (e.g. macrolides) for all patients with CAP who are admitted to hospital after prior treatment with beta-lactam antibiotics. However, whether this protocol will result in improved clinical outcome should be subject to additional study.
The observed reduction in the frequency of S. pneumoniae in patients who received prior outpatient antimicrobial treatment could also be due to a failure to detect the organisms in cultures. This could mask S. pneumoniae as the causative pathogen. However, we believe that this explanation is less plausible, especially since we also used antigen testing to identify the causative pathogen [19]. In addition, such a mechanism can not explain the finding of an increased probability of pneumonia caused by atypical pathogens. To the best of our knowledge, our study is the first to specifically document and quantify the failure of initial outpatient antibiotic treatment as a predictor of the microbial aetiology of CAP.
This study was conducted in a single teaching hospital in The Netherlands, but we believe that the conclusions drawn will apply to other settings. First, the percentage of identified aetiology (64% in this study) is in agreement with that of other studies using similar microbiological techniques [20–22]. Second, our patient characteristics comply to a great extent with a previous nationwide study on prior outpatient antibacterial therapy as a prognostic factor for mortality in patients hospitalised for pneumonia [23]. In that large database study, the percentage of patients hospitalised after initial outpatient antimicrobial treatment was almost identical to that observed in our study (27 vs. 23%, respectively). In addition, age distribution, co-morbidities and the antibiotic utilisation profile of the outpatients were very similar as were the median duration of hospital stay and in-hospital mortality. Unfortunately, due to limited numbers, we were unable to study an association between prior outpatient antimicrobial treatment and mortality in our study. A very reassuring finding was that 85% of all outpatient antibiotic prescriptions complied with national guidelines on the initial treatment of adults with suspected pneumonia. This reduces the possibility that the findings, rather than being associated with antibiotics, might reflect the diagnostic acumen of the physicians who saw the patients in primary care. However, we cannot rule out the possibility that typical signs of infection with atypical pathogens may have been missed by primary care physicians.
As well as finding a relation between prior antimicrobial treatment and aetiology, we also found an association between aetiology and age and pulmonary co-morbidity. Patients aged <60 years without co-morbidities were more likely to have an aetiology comprising viral or atypical bacterial pathogens, and pulmonary co-morbidity was independently associated with S. pneumoniae and H. influenzae as causative pathogens. These findings confirm the results of previous studies on the impact of age and co-morbidity on the microbial aetiology of CAP [6]. A limitation of the present study, however, is that we were not able to adjust for smoking habits and alcohol intake of the patients. Previous studies on determinants for pneumonia aetiology found that these factors are significant predictors of pneumococcal infection [6, 7]. On the other hand, we do not expect prior antimicrobial therapy and smoking and alcohol intake to coincide in such a way that this would result in a null effect when all the information is available.
In conclusion, among patients admitted for pneumonia, whether or not a patient has received prior antimicrobial therapy as an outpatient provides relevant information in the diagnostic workup, in particular in terms of identifying the causative pathogen and planning the initial treatment at the time of hospital admission. This finding supports a further strengthening of the continuity of care at the interface between the extramural and hospitalised settings.

antibiotics
aetiology
pneumonia
diagnosis
outcome assessment


Retrograde traffic in the biosynthetic-secretory route

In the biosynthetic-secretory route from the rough endoplasmic reticulum, across the pre-Golgi intermediate compartments, the Golgi apparatus stacks, trans Golgi network, and post-Golgi organelles, anterograde transport is accompanied and counterbalanced by retrograde traffic of both membranes and contents. In the physiologic dynamics of cells, retrograde flow is necessary for retrieval of molecules that escaped from their compartments of function, for keeping the compartments’ balances, and maintenance of the functional integrities of organelles and compartments along the secretory route, for repeated use of molecules, and molecule repair. Internalized molecules may be transported in retrograde direction along certain sections of the secretory route, and compartments and machineries of the secretory pathway may be misused by toxins. An important example is the toxin of Shigella dysenteriae, which has been shown to travel from the cell surface across endosomes, and the Golgi apparatus en route to the endoplasmic reticulum, and the cytosol, where it exerts its deleterious effects. Most importantly in medical research, knowledge about the retrograde cellular pathways is increasingly being utilized for the development of strategies for targeted delivery of drugs to the interior of cells. Multiple details about the molecular transport machineries involved in retrograde traffic are known; a high number of the molecular constituents have been characterized, and the complicated fine structural architectures of the compartments involved become more and more visible. However, multiple contradictions exist, and already established traffic models again are in question by contradictory results obtained with diverse cell systems, and/or different techniques. Additional problems arise by the fact that the conditions used in the experimental protocols frequently do not reflect the physiologic situations of the cells. Regular and pathologic situations often are intermingled, and experimental treatments by themselves change cell organizations. This review addresses physiologic and pathologic situations, tries to correlate results obtained by different cell biologic techniques, and asks questions, which may be the basis and starting point for further investigations.
Introduction
The biosynthetic-secretory route is traveled by newly synthesized luminal and membrane proteins and glycoproteins from their sites of synthesis at bound ribosomes of the rough endoplasmic reticulum (RER) to their final destinations inside and outside the cells (for review e.g. Farquhar and Hauri 1997; Mellman and Warren 2000). It involves complex and highly dynamic organelles, such as the Golgi apparatus, pre-Golgi intermediates, and post-Golgi organelles, which all have central roles in transport regulation, sorting, and targeting. The route that leads out of the RER at special ER-export sites, involves complicated ER-Golgi intermediate compartments, enters the Golgi apparatus stacks at their cis side, leads across the stacks of Golgi cisternae, where the traversing molecules are subjected to major modifications, and continues at the trans Golgi side. The trans Golgi network (TGN) is a central place of molecule sorting, and packaging of cargo molecules into vehicles for transport to the final intra or extracellular destinations, where the molecules eventually exert their specific functions, such as organelles of the lysosomal system, diverse domains of the plasma membrane, and the extracellular space. At all levels of the biosynthetic-secretory route, anterograde transport of membranes and cargo is counterbalanced by retrograde traffic (for review Sannerud et al. 2003). This is necessary for several reasons, which include the retrieval of molecules that have escaped from the sites of their specific functions, for membrane balance and the maintenance of the functional integrities of organelles and compartments along the secretory route and repeated use of molecules, and molecule repair. Major antero-retrograde traffic cycles are located at the pre-Golgi junction between the ER, the intermediate compartment, and the Golgi apparatus, as well as at the post-Golgi junctions between the TGN, early and late endosomes, and plasma membrane. Within the Golgi apparatus stacks, anterograde transport is assumed to be counterbalanced by retrograde traffic as well. Internalized molecules have been shown to travel along retrograde biosynthetic pathways (e.g. Gonatas et al. 1983; Pavelka et al. 1998; Vetterlein et al. 2002; Volz et al. 1995), and physiologic retrograde routes are misused by harmful substances to reach the sites of their specific toxic effects (e.g. Pelham et al. 1992; Rapak et al. 1997; Sandvig et al. 1991, 1992, 2002; for review Sandvig and van Deurs 2002, 2005). Since the early report in 1992 by Kirsten Sandvig and colleagues, who for the first time showed that along such pathways toxins could be transported en route from the plasma membrane into early compartments of the biosynthetic-secretory system, such retrograde trails of toxins are at the center of cell biologic and medical research.
For both the cell physiologic retrograde routes, and misused pathways, knowledge about molecular machineries, and insights into regulatory mechanisms are increasing rapidly (e.g. Gokool et al. 2007; Mari et al. 2008; Popoff et al. 2007; Wälchli et al. 2008; Yamane et al. 2007; for review Bonifacino and Rojas 2006; Sannerud et al. 2003). In immuno and fine structural analyses, multiple fine details of the compartments, structures and complex architectures involved have been visualized (e.g. Mari et al. 2008; Marsh 2005; Mogelsvang and Howell 2006; Mogelsvang et al. 2004; Pavelka 2007; Pavelka et al. 1998; Vetterlein et al. 2002, 2003). However, there still exists a considerable lack in understanding the mechanisms involved and uncertainties in the interpretations of individual results obtained by molecular biologic and genetic investigations on one hand, and morphologic findings on the other hand. Well known functional processes yet cannot be exactly attributed to concrete defined compartments, and vice versa, the functional implications of multiple structures and architectures need to be clarified. There is a strong need to bring together the biochemical, molecular biological, genetic, morphologic, immuno-cytochemical, and fine structural results.
Retrograde traffic in cell physiology
Anterograde transport in the secretory route is accompanied and counterbalanced by retrograde traffic at each section of the secretory route, including pre- and post-Golgi areas, and presumably at the level of the stacks of Golgi cisternae as well. The diverse retrograde routes and cycles cannot be seen separately. There exist similarities in the mechanisms and machineries, close relationships, and mutual influences.
Retrograde Golgi-to-ER traffic and pre-Golgi circuits
By retrograde Golgi apparatus-to-ER traffic, ER-resident luminal and membrane proteins are retrieved (Munro and Pelham 1987; Semenza et al. 1990), and membrane proteins, including the cargo receptor ERGIC-53 (Appenzeller et al. 1999; Schweizer et al. 1988), and Golgi-resident proteins, such as glycosyltransferases, recycle (Lee et al. 2004; Lippincott-Schwartz et al. 1990; Storrie et al. 1998; Storrie 2005). It is evident that multiple retrograde traffic routes work in parallel in order to retrieve molecules to the ER, and cycle proteins and lipids between ER and Golgi apparatus. The first retrograde Golgi-to-ER pathway described was the KDEL-receptor mediated transport of luminal ER proteins (Lewis and Pelham 1990; Munro and Pelham 1987; Semenza et al. 1990), which following the export out of the ER together with the flow of multiple other proteins destined for transport to other destinations have to be retrieved to their sites of functions in the ER. This traffic, as well as the transport of type-I transmembrane proteins bearing the double-lysin motif signal (Letourneur et al. 1994) is mediated by COPI-coated vesicles (Nickel and Wieland 2002). The retrieval of escaped ER-proteins takes place most efficiently from the ER-Golgi intermediate compartment (ERGIC) but can occur from successive Golgi apparatus compartments up to the TGN as well. Luminal acidification, and calcium concentration in the lumina of the respective compartments have been suggested to be regulatory factors (reviewed in Sannerud et al. 2003).
Furthermore, COPI-independent transport machineries act in Golgi-to-ER retrograde traffic (Johannes and Goud 2000). Such routes are known to involve Rab6 GTPases (Girod et al. 1999; Storrie 2005; White et al. 1999), key regulators of intracellular membrane traffic (Hammer and Wu 2002; Stenmark and Olkkonen 2001; Zerial and McBride 2001). Three different isoforms are known, Rab6A, Rab6A′, an alternatively spliced variant of Rab6A (Echard et al. 2000), and the brain-specific Rab6B (Wanschers et al. 2007). Recently, it has been shown that Rab6A and Rab6A′ perform different non-overlapping functions in cells, and the Rab6A′ isoform is shown mainly regulating the COPI-independent retrograde pathway to the ER (Del Nery et al. 2006). Rab6A GTPases interact with a subunit of the dynein–dynactin complex, and transport compartments move between Golgi apparatus and ER bidirectionally along the microtubules (Matanis et al. 2002; Short et al. 2002). COPI-independent Rab6A-dependent retrograde pathways are used by Golgi apparatus-resident proteins, such as glycosyltransferases, which cycle continuously between the Golgi apparatus and ER (Martinez et al. 1997; Rhee et al. 2005; Storrie 2005; Storrie et al. 1998).
Retrograde traffic within the Golgi apparatus
In the anterograde biosynthetic-secretory traffic, newly synthesized molecules arriving from the ER and ERGIC are taken up into the Golgi apparatus, visit cisternae of the stacks to be modified in well known subsequent steps, and are sorted to different further destinations at the trans side and TGN. Since decades, it is well established that the stacks of Golgi cisternae are subcompartmentalized into separate functional spaces, in each of which defined sets of enzymes are active, and collaborate in performing specific modifications of the molecules that visit the respective cisternae, e.g. perform changes of the sugar chains of the glycoconjugates, such as secretory and plasma membrane glycoproteins, enzymes of the lysosomal system, and lysosomal membrane constituents (Berger 1985; Farquhar and Palade 1998; Glick 2000; Pavelka 1987; Puthenveedu and Linstedt 2005; Rambourg and Clermont 1997; Roth 1997; Storrie 2005). It is assumed that the cisternae of the Golgi apparatus stacks are visited by the molecules to be modified, and there occurs a flow of membranes and contents across the stacks. However, at present, it is not clear, how in the dynamics of the flow the Golgi apparatus subcompartmentalization, which ensures effective glycosylation and other processing in the secretory pathway, is maintained. Conflicting results make interpretations complicated but there exist indications that retrograde transport of membrane constituents, such as the subcompartment-specific glycosyltransferases, is involved. The question how, and even whether, newly synthesized membrane and cargo proteins traverse the stacked Golgi cisternae, is a major point of debate. Several models are discussed, suggesting either an anterograde transport via vesicles, or anterograde flow by progression of the cisternae themselves, or by tubular connections between the cisternae, or by a signal-mediated temporary opening of channels connecting adjacent cisternae (Kartberg et al. 2005; Malhotra and Mayor 2006; Marsh and Howell 2002; Marsh et al. 2004; Mironov et al. 2005; Pelham and Rothman 2000; Puthenveedu and Linstedt 2005; Rodriguez-Boulan and Müsch 2005; Sallese et al. 2006; Storrie 2005, Trucco et al. 2004). Contradictory results also concern the retrograde traffic, which at least in the “cisternae progression model” is required for maintenance of the specific subcompartments. COPI-coated vesicles, also being involved in the retrograde Golgi-to-ER traffic (see the previous chapter), have been proposed as the candidates for retrograde intra-Golgi traffic but the results are conflicting (summerized by Rabouille and Klumperman 2005), and alternative mechanisms are considered as well, such as traffic across tubular connections between the Golgi cisternae (Marsh et al. 2004; Trucco et al. 2004). The retrograde transport and the cycling of Golgi-resident glycosyltransferases between Golgi apparatus and ER have been shown to be functionally connected with the maintenance of the structure of the Golgi apparatus (Starr et al. 2007; Storrie 2005). Recently, it has been shown that the Rab6-binding protein TMF (TATA element modulatory factor), which is involved in the Rab6-dependent retrograde transport processes both from endosomes to the Golgi apparatus, and from the Golgi apparatus to the ER, via its cytoplasmic region is implicated in the retention of N-acetylgalactosaminyltransferase-2 (Yamane et al. 2007).
Retrograde plasma membrane-to-Golgi traffic and post-Golgi circuits
In part comparable with the pre-Golgi retrograde routes, post-Golgi retrograde traffic on one hand represents a backward flow that counterbalances forward flow by anterograde traffic to the final destinations of newly synthesized molecules to the plasma membrane, extracellular space, secretory granules, endosomes, or lysosomes. Post-Golgi retrograde trafficking concerns TGN-resident proteins, such as TGN38, and receptors involved in the sorting of newly synthesized molecules of the trans Golgi apparatus and TGN to their specific sites of action. This transport is bidirectional; the receptors recycle to the TGN to be reused in further rounds, possibly also being modified or repaired (Bonifacino and Rojas 2006; Rohn et al. 2000; Snyder and Rogers 1985; Volz et al. 1995). Best-studied examples are the cation-independent and the cation-dependent mannose-6-phosphate receptors (CI- and CD-MPR; Ghosh et al. 2003). Other recycling proteins are the multi-ligand receptor sortilin (Mari et al. 2008), TGN38 (Banting and Ponnambalam 1997; Ghosh et al. 1998; Stanley and Howell 1993), GPP130 and GP73 (Puri et al. 2002), processing enzymes, such as the transmembrane endoproteases furin and carboxypeptidase D (Molloy et al. 1999; Varlamov and Fricker 1998), and SNAREs (soluble N-maleimide-sensitive fusion protein /NFS/ attachment protein receptor; Hettema et al. 2003; Hong 2005). At least, two independent retrograde routes from early endosomes to the TGN exist involving specific components of the Rab and SNARE machineries. One leads to the TGN via late endosomes (Barbero et al. 2002; Carrol et al. 2001; Lombardi et al. 1993; Mallet and Maxfiled 1999); the other one is a direct route from early and/or recycling endosomes to the TGN, thus bypassing late endosomes (Mallard et al. 1998, 2002). During the past years, insights into the regulatory mechanisms of retrograde traffic from endosomes to the TGN considerably increased. Multiple constituents of the molecular transport machineries have been characterized and their roles specified; these include Rab9 and TIP47 (tail-interacting protein 47 kDa; Diaz et al. 1997), PACS1 (phosphofurin acidic cluster sorting protein 1) and AP-1 (Crump et al. 2001; Meyer et al. 2000), EpsinR (Saint-Pol et al. 2004), the t-SNAREs syntaxin 16 and 5 (Amessou et al. 2007), and constituents of the retromer complex with the two subcomplexes, a membrane-bound coat that consists of the phosphinositide binding proteins Sorting nexin 1 and possibly Sorting nexin 2, and the cargo-binding proteins Vps26, Vps29, and Vps35 (Arighi et al. 2004; Gokool et al. 2007; Popoff et al. 2007; Restrepo et al. 2007; Rojas et al 2007; Seaman et al. 1998; Seaman 2004, 2005; for review Bonifacino and Rojas 2006). Clathrin and the retromer complex are suggested to function in consecutive retrograde sorting steps on early endosomes (Popoff et al. 2007).
There exist several different carrier compartments for retrograde endosome-to-TGN traffic that can form at the same vacuolar early endosome (Mari et al. 2008): tubular sorting endosomes (Peden et al. 2004), and tubular endosomal networks (Bonifacino and Rojas 2006), and the endosome-to-TGN carriers (Mari et al. 2008). The tubular endosomal network (TEN) is connected to vacuolar early endosomes, which exhibit bilayered coats composed of clathrin and Hrs (hepatocyte-growth-factor-regulated tyrosine kinase substrate) thought to contain the ESCRT machinery that targets proteins to the intraluminal vesicles, thus being sorted to the multivesicular-late endosomal-lysosomal route (Hurley and Emr 2006; Sachse et al. 2002). The vacuolar endosomal part is suggested to receive endocytic cargo from the plasma membrane on one hand, and biosynthetic cargo, e.g. lysosomal enzymes, from the TGN on the other hand; it progressively becomes acidic, leading to the release of the cargo from the receptors. It is proposed that the vacuolar endosomal region matures into late endosomes, whereas from the TEN-region, cargos are sorted to different destination, such as to the TGN and Golgi apparatus along the retrograde route but also to different regions of the plasma membrane for recycling and transcytosis, and to specialized storage compartments, such as melanosomes. The TEN is equipped with different transport machineries including the retromer. It is considered that the pericentriolar endocytic recycling compartment (Ghosh et al. 1998; Maxfield and McGraw 2004; Ullrich et al. 1996) could be a specialized subdomain of TEN. With respect to its central sorting role, TEN is proposed to represent a “mirror image” of the TGN (Bonifacino and Rojas 2006). A possibly comparable endocytic trans-Golgi network has been shown to develop after internalization of wheat germ agglutinin (WGA; Pavelka et al. 1998; Vetterlein et al. 2002; Figs. 1, 2). Recently, a novel class of carriers, the endosome-to-TGN carriers (ETCs), has been characterized (Mari et al. 2008). These carriers transport mannose-6-phosphate receptors and sortilin; they are dependent on the presence of sorting nexin 1 (SNX1), and show unique structures appearing as non-branched tubules and vesicles, clearly different from the tubular sorting endosomes (Peden et al. 2004) and tubular networks (Bonifacino and Rojas 2006).
Fig. 1The Golgi apparatus reorganizes during endocytosis of WGA within a period of 30 min. ainsert: The insert shows WGA-HRP reaction products concentrated in a clathrin-coated pit, and a coated vesicle (arrows), as well as lining the membrane of a small uncoated vesicle (arrowhead). ×35,000. a Small endosomes (arrowheads), and a large vacuolar endosome (arrow) are apparent in the cytocentre (c), and close to a small Golgi apparatus stack (Golgi). A distinct TGN is not visible. ×30,000. b Globular early endosomes are accumulated close to the trans side of Golgi apparatus stacks (Golgi); some of the endosomes are covered with clathrin coats (arrowheads); partly, they exhibit WGA reaction products attached to the membranes, partly contained within the lumina. Some of endosomes appear to contact each other, and fine bridges are visible (short arrows). WGA reactions are apparent within the cisternae of a small Golgi apparatus stack (large arrow). ×31.500. c A network, an endocytic trans Golgi network (endocytic TGN), is apparent consisting of interconnected globular pieces (arrowheads) that resemble the earlier globular endosomes. Again here, the WGA reaction products are either attached to the membranes, or fill the lumina. Parts of this endocytic TGN are attached to stacks of Golgi cisternae at their trans sides (arrows), and are associated with trans Golgi ER. At the cis side, ER-Golgi-intermediate compartments (ERGIC) are visible. ×28,000. d Endocytic TGN consisting of interconnected globular pieces (arrowheads) and filled with WGA reaction products are partly attached to Golgi apparatus stacks (arrows), and associated with trans Golgi ER. ×32,000Fig. 2Portions of the endocytic TGN are integrated in the Golgi apparatus stacks a The WGA-labeled endocytic TGN (long arrows) represents an integrated part of this Golgi apparatus stack, and is closely associated to the transmost Golgi cisterna at one side, and with trans Golgi ER at the opposite side. One large vacuolar part (V) of the endocytic TGN is visible; some of the globular pieces of the endocytic TGN are covered with clathrin coats (short arrow). ×35,000. b The endocytic TGN (arrows) consists of globular pieces, and a large vacuole part (V), which here is attached to the Golgi apparatus stack. ×44,000Fig. 3a–d This 3D-model of a Golgi apparatus stack containing integrated endocytic TGN, has been formed according to the data of an electron tomography tilting series. The model is shown from different sides. The endocytic TGN (green) consists of a free portion (long arrows), and another portion integrated in the stack (short arrows), and is closely associated with trans Golgi ER (red). In both the free and the Golgi-associated portions, globular elements are visible. Stacked Golgi cisternae are shown in yellow. In c, the stacked Golgi cisternae are removed. Spikes mark clathrin coats
Retrograde traffic of internalized molecules
The plasma membrane-endosome-TGN pathway used by plasma membrane proteins for reentry into the biosynthetic system for reuse, or modification and repair (Snyder and Rogers 1985; Volz et al. 1995; reviewed in Bonifacino and Rojas 2006) also opens the secretory pathway for entry of extracellular ligands, including harmful substances, such as bacterial and plant toxins (for review Sandvig and van Deurs 2005), and provides tracks for drug delivery to the interior of cells (for review Tarrago-Trani and Storrie 2007).
Retrograde routes of lectins and toxins
Since more than two decades, it is known that lectins, and bacterial and plant toxins, such as the Shiga and Cholera toxins, the Pseudomonas exotoxin A, and ricin, utilize the retrograde routes within the cells to travel to the TGN and Golgi apparatus, ER, and cytosol (e.g. Gonatas et al. 1983, Sandvig and Brown 1987; van Deurs et al. 1986, 1987). Sandvig et al. (1992) were the first, who showed that Shiga toxin is transported from the cell surface en route to the endoplasmic reticulum. Shiga toxin consists of an enzymatically active A-subunit that is noncovalently linked to a pentamer of B-chains. The latter binds to the glycosphingolipid Gb3, and mediates the toxin’s transport. Following internalization, the toxin is transported to early endosomes, sorted to the TGN and Golgi apparatus, and further transported to the ER, from where after cleavage the A-subunit is retrotranslocated to the cytosol, the site where it exerts its toxic action by inactivation of ribosomes, and inhibition of protein synthesis (Johannes and Goud 1998, 2000; Sandvig and van Deurs 1994, 1996, 2002, 2005). It is well established that different uptake mechanisms are used, and there exists not only one route to the endoplasmic reticulum but different retrograde pathways may be traveled by different toxins, and trafficking of one class of toxin is not limited to one route, e.g. Pseudomonas exotoxin A has been shown to travel to the endoplasmic reticulum along multiple pathways (Smith et al. 2006). There also appears to be fundamental differences between the endosomal sorting into the retrograde pathway to the TGN of Shiga and Cholera toxins (Bujny et al. 2007; Chinnapen et al. 2007; Feng et al. 2004; Massol et al. 2004; Torgersen et al. 2001). Multiple details concerning different uptake mechanisms, and retrograde traffic of Shiga toxin and ricin have been elucidated during the past years (e.g. Amessou et al. 2007; Garred et al. 1995; Grimmer et al. 2005, 2006; Johannes et al. 1997; Lauvrak et al. 2004, 2006; Mallard et al. 1998; Rapak et al. 1997; Römer et al. 2007; Skanland et al. 2007; Slominska-Wojewodzka et al. 2006; Tai et al. 2004; Torgersen et al. 2007; Utskarpen et al. 2006, 2007; Yoshino et al. 2005). A key compartment is the early endosome (see previous chapter), from where internalized molecules, are either recycled to the plasma membrane to be reused, or sorted to the late endosomal pathway and to the lysosomes to be degraded, or transferred to a direct pathway to the TGN, travelled by endogenous proteins, like the mannose-6-phosphate receptors (for review Bonifacino and Rojas 2006). It is the latter route that is travelled by Shiga toxin and ricin. For efficient transport of Shiga toxin from early endosomes to the TGN and Golgi apparatus, the retromer complex is required (Bujny et al. 2007; Popoff et al. 2007; Utskarpen et al. 2007). The ricin endosome-to-TGN and Golgi traffic has been shown to be facilitated by depletion of sphingolipid (Grimmer et al. 2006), and to be dependent on Rab6A and Rab6A′ (Utskarpen et al. 2006), which are also regulators in pre-Golgi circuits (see previous chapter). The syntaxins 5 and 16 being involved in retrograde transport of mannose-6-phosphate receptors, also are necessary for efficient retrograde traffic of Shiga toxin, as well as for trafficking into the cells of ricin and cholera toxin (Amessou et al. 2007). In the center of interest are the regulatory roles of phosphoinositides (for review De Matteis and Godi 2004), and the importance of signaling (Pelkmans et al. 2005; Perret et al. 2005; von Zastrow and Sorkin 2007). Recently, it has been demonstrated that retrograde traffic of Shiga toxin and ricin are phosphoinositide-regulated (Skanland et al. 2007; Utskarpen et al. 2007). The phosphoinositide-binding proteins sorting nexins 1 and 2, being part of the retromer complex (Seaman 2004, 2005), are necessary for efficient transport of Shiga toxin to the Golgi apparatus (Bujny et al. 2007; Popoff et al. 2007; Utskarpen et al. 2007). Sorting nexins have been shown to be in crosstalk with the phosphatidylinositol (PI) 3-kinase hVps34; it is proposed that hVps34 produces a specific PI(3)P pool needed for the localization of sorting nexins on endosome vesicles, which in turn is required for retrograde endosome-to-Golgi traffic of ricin (Skanland et al. 2007).
Shiga toxin is an active player in its own transport mediating both internalization, and intracellular transport. Upon binding to the plasma membrane or entry into the cells, it is able to trigger signaling cascades (Ikeda et al. 2000; Lauvrak et al. 2006; Wälchli et al. 2008). Shiga toxin activates the tyrosine kinase Syk, by which clathrin phosphorylation and uptake of Shiga toxin is induced (Lauvrak et al. 2006). Protein kinase Cδ is specifically activated by Shiga toxin regulating endosome-to-Golgi transport (Torgersen et al. 2007). Evidence is provided for activation of a signaling cascade that involves a crosstalk between Ca2+ and the MAP kinase p38; it is suggested that Shiga toxin, by modifying Ca2+ homeostasis, recruits p38 to endosomes for regulation of transport to the Golgi apparatus (Wälchli et al. 2008).
The entry of internalized molecules into the secretory pathway at the TGN and Golgi apparatus level is a major event influencing further retrograde traffic to the ER and cytosol. Although it is well established, and has been shown ultrastructurally very early for several toxins, that transport to the TGN and Golgi apparatus is followed by uptake into cisternae of the Golgi apparatus stacks (e.g. Sandvig et al. 1992; van Deurs et al. 1987), the involved mechanisms are still poorly understood. A detailed ultrastructural analysis has been undertaken using a HepG2 hepatoma cell model, and uptake of wheat germ agglutinin labeled with horseradish peroxidase (WGA-HRP; Pavelka et al. 1998; Pavelka 2007; Vetterlein et al. 2002).
Wheat germ agglutinin is an N-acetyl-glucosamine and sialic acid-specific lectin, which is known for many years to be transported to the Golgi apparatus in retrograde direction (e.g. Gonatas et al. 1977; Stieber et al. 1984); it is also used in connection with the developments of drug delivery systems (Lochner et al. 2003; Weissenböck et al. 2004). WGA reacts with numerous binding sites at the cell surface, and is taken up in large amounts, thus mimicking normal situations in cells, and here particularly reflecting the functions of hepatocytes in the liver tissue. HepG2 hepatoma cells are especially well suited for these studies, since prior to, and in the initial phases of WGA endocytosis, the Golgi apparatus mainly consists of small inconspicuous stacks of cisternae (Fig. 1a), and the changes and reorganizations of the Golgi apparatus during WGA-endocytosis are clearly visible (Figs. 1, 2, 4). Briefly, the results show that internalized WGA, following internalization via clathrin-coated vesicles (Fig. 1a insert) and possibly other mechanisms as well, is rapidly transported to the Golgi apparatus, and induces dramatic Golgi reorganizations. Early globular endosomes accumulate at the trans Golgi side (Fig. 1b) and a network is formed, an endocytic trans-Golgi network (endocytic TGN) that consists of interconnected globular pieces (Fig. 1c), which in dimensions and shapes resemble the earlier globular endosomes (Fig. 1b). In these compartments, the WGA reaction products in part detach from the limiting membranes, and fill the lumina (Fig. 1b, c, d) indicating that the luminal milieu is changing. Portions of the endocytic TGN in close association with trans-Golgi ER, attach to trans Golgi cisternae (Fig. 1c, d), thus becoming integrated parts of the Golgi apparatus stacks (Figs. 2a, b, 4a; various views of a 3D-model are presented in Fig. 3a–d). The trans-Golgi attachment of the endocytic TGN leads to interconnections of the small Golgi stacks (Figs. 1c, d, 4b), and causes the formation of Golgi apparatus ribbons. Concomitantly, and in part prior to the formation of an endocytic TGN, internalized WGA appears within cisternae of Golgi apparatus stacks (Figs. 1b, d, 4a). Stacks that contain high amounts of WGA within the lumina of all the cisternae are mainly found at later periods of WGA endocytosis, e.g. after 45–60 min (Fig. 4b). Very seldom, internalized WGA can be localized within the ER cisternae. At all periods of WGA endocytosis, internalized WGA appears within multivesiculated bodies (Fig. 4a, b), indicating that internalized WGA not only trafficks into the Golgi apparatus but is sorted to the late endosomal–lysosomal pathway as well.
Fig. 4WGA is taken up into the stacked Golgi cisternae a Free portions (long arrows), and Golgi-integrated portions (short arrows) of the WGA-reactive endocytic TGN are visible. Fine WGA reactions also are apparent at confined regions of some of the stacked Golgi cisternae (double arrow). Several multivesiculated bodies (MVB) are densely filled with WGA-reaction products, and show domains, where tubular and vesicular transport carriers are formed. ×20,000. b A Golgi ribbon is visible, in which the individual stacks of cisternae are interconnected at the cis, as well as at the trans side. Most of the stacked Golgi cisternae are densely filled with WGA reaction products; others show reactions at limited regions (arrow). At the right lower corner, connections of medial cisternae with the cis-most cisterna of the same stack, and of other stacks are shown. In a vacuolar endosome (V), WGA reaction products are attached to the limiting membrane, and to an intra-vacuolar vesicle (arrowhead). ×23,000
The detailed knowledge of these endocytosis-induced Golgi reorganizations has been used to develop a precise time schedule for regulated retrograde transport of WGA into the endoplasmic reticulum by treatment with Brefeldin A (Vetterlein et al. 2003). However, multiple questions are open, which concern the mechanisms of formation of the endocytic TGN, and its attachment to the Golgi stacks, the delivery of cargo, and uptake of membranes, interactions of transport carriers with Golgi subcompartments, mechanisms of retrograde transport within the Golgi apparatus stacks, signaling and the role of contact points, the importance of lipid transfer (De Matteis et al. 2007; Hanada et al. 2007), and possible direct endocytic TGN-to-ER traffic via trans-Golgi associated ER. Of particular interest are questions, as to whether secretory anterograde flow influences retrograde traffic, and vice versa, as to whether retrograde flow influences Golgi apparatus size and localization, and whether the formation of Golgi ribbons by retrograde flow is comparable with the formation of Golgi ribbons by input of membrane derived from the ER (Marra et al. 2007).
Concluding remarks
In future studies, it will be important to correlate the detailed fine structural findings with the molecular biologic and genetic results on the machineries and regulatory mechanisms of retrograde traffic (e.g. Amessou et al. 2007; Bonifacino and Rojas 2006; De Matteis et al. 2007; Hanada et al. 2007; Levine and Loewen 2006; Marra et al. 2007; Missiaen et al. 2007; von Zastrow and Sorkin. 2007; Wälchli et al. 2008). The crucial roles of retrograde traffic in the biosynthetic-secretory route for cellular homeostasis and intoxication of cells, the importance for assessment of effects and side effects of drugs (e.g. Sandoval and Molitoris 2004), and the development of strategies for targeted drug delivery to the interior of cells (El Alaoui et al. 2007; Johannes and Decaudin 2005; Kreitman 2006; Smith et al. 2002; Tarrago-Trani and Storrie 2007; Weissenböck et al. 2004) will be the driving forces.

retrograde traffic
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golgi apparatus
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biosynthetic secretory route