The distal fascicle of the anterior inferior tibiofibular ligament as a cause of tibiotalar impingement syndrome : a current concepts review Impingement syndromes of the ankle involve either osseous or soft tissue impingement and can be anterior , anterolateral , or posterior . Ankle impingement syndromes are painful conditions caused by the friction of joint tissues , which are both the cause and the effect of altered joint biomechanics . The distal fascicle of the anterior inferior tibiofibular ligament -LRB- AITFL -RRB- is possible cause of anterior impingement . The objective of this article was to review the literature concerning the anatomy , pathogenesis , symptoms and treatment of the AITFL impingement and finally to formulate treatment recommendations . The AITFL starts from the distal tibia , 5 mm in average above the articular surface , and descends obliquely between the adjacent margins of the tibia and fibula , anterior to the syndesmosis to the anterior aspect of the lateral malleolus . The incidence of the accessory fascicle differs very widely in the several studies . The presence of the distal fascicle of the AITFL and also the contact with the anterolateral talus is probably a normal finding . It may become pathological , due to anatomical variations and/or anterolateral instability of the ankle resulting from an anterior talofibular ligament injury . When observed during an ankle arthroscopy , the surgeon should look for the criteria described to decide whether it is pathological and considering resection of the distal fascicle . The presence of the AITFL and the contact with the talus is a normal finding . An impingement of the AITFL can result from an anatomical variant or anteroposterior instability of the ankle . The diagnosis of ligamentous impingement in the anterior aspect of the ankle should be considered in patients who have chronic ankle pain in the anterolateral aspect of the ankle after an inversion injury and have a stable ankle , normal plain radiographs , and isolated point tenderness on the anterolateral aspect of the talar dome and in the anteroinferior tibiofibular ligament . The impingement syndrome can be treated arthroscopically . Introduction Impingement syndromes of the ankle involve either osseous or soft tissue impingement and can be anterior , anterolateral , or posterior -LSB- 5 , 25 -RSB- . Ankle impingement syndromes are painful conditions caused by the friction of joint tissues , which are both the cause and the effect of altered joint biomechanics -LSB- 14 -RSB- . The leading causes of impingement lesions are post-traumatic ankle injuries , usually ankle sprains , resulting in chronic ankle pain -LSB- 33 -RSB- . According to the ISAKOS definition , anterior ankle impingement is a pain syndrome characterised by anterior ankle pain on activity . Recurrent dorsiflexion is often the cause . On investigation , there is pain on palpation at the antero/medial and/or anterolateral aspect of the ankle joint . Some swelling and/or limitations of dorsiflexion are present -LSB- 32 -RSB- . The first reported impingement syndromes about the ankle have evolved osseous impingement and generally have been noted in athletes whose sports necessitated sudden acceleration , jumping , and extremes of dorsiflexion or plantar flexion . Impingement of the ankle was first described in the English language literature by Morris -LSB- 20 -RSB- , in 1943 , in five patients who had what he called the athlete 's ankle . This was later called footballer 's ankle in the report of McMurray -LSB- 18 -RSB- . Soft tissue impingement of the ankle is a common cause of chronic ankle pain that usually arises at the lateral and/or anterolateral compartment of the ankle joint following an inversion injury -LSB- 8 -RSB- . It is estimated that approximately 3 % of ankle sprains may lead to anterolateral impingement -LSB- 32 -RSB- . Three types of intra-articular soft tissue lesions that lead to these complaints have been described : the meniscoid lesion , synovitis , and the distal fascicle of the anterior inferior tibiofibular ligament -LRB- AITFL -RRB- -LSB- 4 , 6 , 8 , 9 , 12 , 16 , 19 , 28 -RSB- . The formation of tibiotalar spurs due to repetitive capsule traction therefore does not seem plausible reason for anterior ankle impingement -LSB- 29 -RSB- . Impinging soft tissue pathology in the ankle was first described by Wolin et al. as a `` meniscoid mass or lesion '' -LSB- 34 -RSB- . They described the lesion as a membrane shaped hyalinised scar tissue obstructing the lateral talofibular articulation in nine patients with persistent symptoms over the anterolateral corner of the ankle after an inversion trauma -LSB- 34 -RSB- . Some authors thought this lesion was formed from the torn ends of the anterior tibiofibular ligament -LSB- 3 , 21 -RSB- . However there was no ligamentous tissue found on histologic examination of this lesion -LSB- 9 -RSB- . Guhl thought the lesion was of synovial origin -LSB- 11 -RSB- . Irritation of the joint by bleeding and a torn capsule or a torn anterior tibiofibular ligament are thought to be the underlying factors for developing post-traumatic synovitis in the ankle -LSB- 17 -RSB- which in unstable ankles may lead to formation of fibrosis and granulation tissue . Although synovitis and the meniscoid lesion have been referred frequently , impingement by a separated fascicle of the AITFL is a relatively new and unknown pathology . Bassett et al. -LSB- 4 -RSB- were the first who reported a ligamentous etiology of impingement of the anterior aspect of the ankle . The presence of the so-called accessory AITFL , as described by Nikolopoulos was confirmed by them -LSB- 21 -RSB- . However they called this accessory ligament , the distal fascicle of the normal AITFL -LSB- 4 -RSB- . The distal part of the fascicle rubs against the talus , and that this causes pain at the ankle -LSB- 1 , 2 , 4 , 8 , 12 , 22 -RSB- . The objective of this article was to review the literature concerning the anatomy , pathogenesis , symptoms and treatment of the AITFL impingement and finally to formulate treatment recommendations . Anatomy of the anterior inferior tibiofibular ligament The distal tibiofibular joint was classically described as a syndesmosis between the rough , triangular surface on the medial and distal aspect of the fibular shaft and the fibular notch of the distal end of the tibia . The tibiofibular syndesmosis is established by three ligaments : the interosseous ligament , the posterior inferior tibiofibular ligament , and the AITFL -LSB- 7 , 10 , 15 , 24 , 26 -RSB- . The AITFL is a flat band becoming thicker from superior to inferior -LSB- 24 -RSB- . The ligament starts from the distal tibia , 5 mm in average above the articular surface , and descends obliquely between the adjacent margins of the tibia and fibula , anterior to the syndesmosis to the anterior aspect of the lateral malleolus -LSB- 7 , 22 -RSB- . Its length ranges from 12 to 20 mm -LSB- 4 , 22 -RSB- ; its thickness , from 1 to 3 mm -LSB- 4 , 22 -RSB- ; and its width ranges from 7 to 12 mm at the fibular insertion and 9 to 22 mm at the tibial insertion -LSB- 22 -RSB- . This ligament is the weakest of the syndesmotic ligaments and is the first to yield to forces that promote an external rotation of the fibula around its longitudinal axis -LSB- 15 -RSB- . Resection of the accessory ligament did not disturb the stability of the syndesmosis , a finding that was in accordance with the results reported by Rasmussen et al. which suggests that mobility is minimally influenced by isolated cutting of the AITFL -LSB- 23 -RSB- . Nikolopoulos et al. studied 24 cadaveric ankles to describe the anatomy of the AITFL -LSB- 22 -RSB- . In five ankles the AITFL appeared to consist of two layers , one superficial and one deep , which were separated by a thin fibrofatty septum -LSB- 22 , 24 -RSB- . However , the total thickness of these two layers did not exceed the average thickness of the AITFL in the rest of the specimens -LSB- 22 -RSB- . In five ankles , the articulated surfaces of the distal tibia and fibula that formed the syndesmosis were covered by articular cartilage and were surrounded by synovial bursa -LSB- 22 -RSB- . A completely separate accessory anteroinferior tibiofibular ligament was present in 22 ankles -LRB- Fig. 1 -RRB- . Nikolopoulos was the first to describe this accessory ligament -LSB- 21 -RSB- . However the results of his study were never published in English literature -LSB- 26 -RSB- . Nikolopoulos believed that this anatomical structure represents a separate ligament rather than a distal component of the AITFL -LSB- 21 -RSB- . The fact that there was clearly a fibrofatty septum that separates the AITFL from the accessory fascicle , similar to the one that covered the intermediate space between the interosseous and the anterior or the interosseous and the posterior tibiofibular ligaments , suggested that this fascicle could probably be considered an independent accessory ligament -LSB- 22 -RSB- . Some other authors described this accessory ligament as a complete separate distal fascicle of the AITFL -LSB- 4 , 15 , 26 -RSB- . Fig. 1The distal fascicle in relation to the anterior inferior tibiofibular ligament Ray and Kriz -LSB- 24 -RSB- founded in 10 of the 46 specimens an accessory ligament . This ligament was parallel and distal to the AITFL . The fibres of the accessory ligament descended obliquely from the lateral , anterior , and distal borders of the tibia to the anteromedial aspect of the lateral malleolus , approximating the fibular insertion of the anterior talofibular ligament -LRB- ATFL -RRB- . Its length ranges from 17 to 22 mm , its thickness from 1 to 2 mm , and its width from 3 to 5 mm . The maximum , minimum , mean , and standard deviation for the length , width , gap , and angle of declination for the inferior fibre bundle are provided in Table 1 . The ligament was not covered by synovial tissue , ran deeper than the AITFL and was described as being intra-articular , although how this was determined was not stated by the authors . The accessory ligament crossed the superior lateral margin of the ankle joint . The accessory ligament was separated from the AITFL by a distinct fibrofatty septum . The ligament could even be divided in two , three , or more fascicles , increasing in length from superior to inferior -LSB- 24 -RSB- . Table 1Characteristics of the inferior fascicle of the AITFL -LSB- 24 -RSB- MaximumMinimumMeanLigament length -LRB- cm -RRB- 3.42.12.6 ± 0.3 Ligament width -LRB- cm -RRB- 1.30.20.4 ± 0.2 Gap -LRB- cm -RRB- 0.50.10.2 ± 0.1 Declination angle -LRB- deg -RRB- 155.0121.5136.3 ± 7.4 The incidence of the accessory fascicle differs very widely in the several studies -LRB- 21 -- 92 % -RRB- -LSB- 2 , 4 , 22 , 24 -RSB- . The discrepancies in the incidence between the several studies were probably due to the different definitions of a separate fascicle . Ray and Kriz did not consider it a separate fascicle if any digitations were present between the fascicle and the main body of the AITFL -LSB- 24 -RSB- . The incidence was up to 92 % , which implied that the ligament may be considered as a normal finding . Vessels from the anterior peroneal artery penetrate through the interlaminar spaces -LSB- 26 -RSB- . It is not uncommon to have branches from the anterior tibial artery or peroneal artery or a branch arising from the anastomosis between these two arteries pass between these fascicles -LSB- 11 -RSB- . The most inferior fibres of the ligament merged at their fibular attachment with the origin of the ATFL -LSB- 24 -RSB- . Ray and Kriz -LSB- 24 -RSB- studied 23 cadaveric ankles . A classification scheme was designed to categorise the anatomical variations observed in the AITFL -LRB- Table 2 -RRB- . Table 2AITFL classification system -LSB- 24 -RSB- Type 1Multiple fascicles -LRB- more than three -RRB- with or without small gaps between adjacent fascicles 1AThe inferior fascicle is separated from the rest of the ligament by a gap and possesses its own distinct proximal and distal attachments 1BThe inferior fascicle is not completely separated from the rest of the ligament by a gap . Either its proximal or its distal attachment is continuous with the rest of the ligament 1CMultiple fascicles without gaps intervening between themType 2Three fascicles or less . A distinct inferior fascicle with both its proximal and distal attachments separate from the rest of the ligament . The inferior fascicle is separated completely from the main portion of the ligament by a gapType 3Three fascicles or less . A distinct inferior fascicle with either its proximal or its distal attachment continuous with the rest of the ligament . A gap does not completely separate the inferior fascicle from the rest of the ligamentType 4Three fascicles or less . The lower portion of the ligament possesses an inferior fascicle with both its proximal and distal attachments for the rest of the ligamentType 5Three fascicles or less . A ligament with no separations or gaps within its structure . It may or may not possess a fascicular arrangement Pathomechanism of the impingement syndrome It should be stressed that most studies were carried out in vitro and do not take muscular tonus and the effects of weightbearing into consideration , we performed a review of the pathomechanism of the impingement syndrome caused by the distal fascicle of the AITFL . Bassett et al. correlated the presence of this accessory ligament with pain from impingement in the anterolateral aspect of the ankle in patients with a history of inversion sprains -LSB- 4 -RSB- . They postulated that the post-traumatic anterolateral hyperlaxity , due to an injured ATFL , resulted in anterior extrusion of the talar dome with dorsiflexion , which now contacted the inferior fascicle of the AITFL with more pressure and friction -LSB- 4 -RSB- . This in turn might set up an environment favouring ligamentous impingement of the anterior aspect of the ankle -LSB- 4 -RSB- . Akseki et al. stated that the variations in width , length , and obliquity of the fascicle may be related to the pathological behaviour of the fascicle -LSB- 2 -RSB- . Mean width and length of the fascicle with bending during dorsiflexion and dorsiflexion-eversion were significantly higher than the fascicle without bending . Therefore this wider and longer fascicles had more potential to become pathological than thinner ones . They also conclude that if the fibular insertion point is far from the joint level , the fascicle has more potential to become pathological . These findings might be useful during arthroscopic procedures of the ankle . During dorsiflexion of the ankle the posterior shift of the anterior talus below the tibial plafond in ATFL intact cases was observed . Following incision of the ATFL this posterior shift of the talus was not observed ; the talus remained in an anterior position and thus in contact with the fascicle -LSB- 2 -RSB- . Lesion of the ATFL led to bending of the fascicle with dorsiflexion and dorsiflexion-inversion in the specimens which the bending sign was negative . Lesion of the ATFL led also to contact between the talus and the fascicle in the ankles which showed no contact previously -LSB- 2 -RSB- . After sectioning the ATFL the anteroposterior laxity of the ankle increases especially in dorsiflexion -LSB- 4 , 13 -RSB- . In 89.3 % of the cases contact was observed between the ATFL and the anterolateral corner of the talus -LSB- 2 -RSB- . This contact may be normal in some of the cases . Contact was observed at an average of 12 ° of dorsiflexion of the ankle -LSB- 4 -RSB- . Bending of the accessory ligament , indicating further tensioning of its ligamentous bands caused by impingement of the talus , this was noted in an extent of dorsiflexion that varied from 5 ° to 10 ° -LRB- average 7 ° -RRB- -LSB- 22 -RSB- . Maximum dorsiflexion of the foot relieved the contact between the ligament and the superolateral corner of the talus -LSB- 22 -RSB- . In other studies the contact was also observed in neutral position -LSB- 2 , 24 -RSB- . In one study the contact between the fascicle and talar dome was observed throughout the whole range of motion of the ankle -LSB- 1 -RSB- . They called this the `` arthroscopic impingement test , '' and it was positive in all of the cases with AITFL impingement they have studied -LSB- 1 -RSB- . Contact between the accessory fascicle and the talus was also observed during eversion of the foot , being more prominent when the foot was placed in a dorsiflexion-eversion position -LSB- 22 -RSB- . No interaction of the accessory ligament with the talus was noted during ankle plantar flexion -LSB- 22 -RSB- . In 82.9 % of the cases with AITFL impingement Ray and Kriz -LSB- 24 -RSB- found an anterior abraded articular region of the talus . Akseki et al. -LSB- 2 -RSB- found this in 17 % of the cases . In the study of Basset et al. -LSB- 4 -RSB- 71 % of the patients , had an area of abraded articular cartilage on the anterolateral surface of the talar dome . There is no clarity if this abraded cartilage region is an anatomical variant of the talus or a pathological remodelling of the bone due to true ligamentous impingement -LSB- 4 -RSB- . On base of these studies we conclude that anterolateral hyperlaxity results in anterior extrusion of the talar dome with dorsiflexion , which contacts the inferior fascicle of the AITFL with more pressure and friction . This hyperlaxity was most likely due to an injured ATFL . Wider and longer fascicles and a fibular insertion point far from the joint level have more potential to become pathological . Often contact between the AITFL and the superolateral corner of the talus and an abraded region of the cartilage of the talus can be observed during arthroscopy . Diagnosis The diagnosis of ligamentous impingement in the anterior aspect of the ankle should be considered in patients who have chronic ankle pain in the anterolateral aspect of the ankle after an inversion sprain and who have a stable ankle , normal plain radiographs , and isolated point tenderness on the anterolateral aspect of the talar dome and in the anteroinferior tibiofibular ligament -LSB- 4 -RSB- . A popping sensation and aggravation of pain with dorsiflexion and eversion were noted -LSB- 2 , 4 -RSB- . This audible popping is usually not seen in other causes of impingement of the anterior aspect of the ankle -LSB- 4 -RSB- . Pain in the beginning of plantarflexion-inversion may alert the physician for an impinging AITFL -LSB- 2 -RSB- . It is usually impossible to distinguish the source of pain in patients with both instability and impingement . So if the patient complains of giving way and frequent sprains , the surgeon may choose a reconstructive procedure -LSB- 1 -RSB- . But it should be noted that a painful lesion in the joint -LRB- synovitis , meniscoid lesion , or an impinging fascicle of the AITFL -RRB- may cause reflex sprains -LSB- 1 -RSB- . This process may confuse decision making in this type of combined pathology . The differential diagnosis for pain in the lateral aspect of the ankle after an inversion sprain includes osteochondral fracture , instability of the lateral aspect of the ankle , synovitis or rupture of the peroneal tendon , and ligamentous impingement in the anterior aspect of the ankle , with the latter being the least common -LSB- 4 -RSB- . Treatment Patients should be treated conservatively after an inversion sprain of the ankle for at least 6 months before operative intervention is considered -LSB- 5 -RSB- . This is with the exception of patients with an osteochondral fracture . Akseki et al. observed that conservative management with physical therapy , NSAIDs and bracing in patients with instability symptoms , for a period of 3 months , failed in all 21 patients -LSB- 1 -RSB- . Not every patient with chronic ankle instability needs a reconstructive procedure -LSB- 1 -RSB- . If the history , physical examination , and other diagnostic tests suggest impingement in addition to instability , arthroscopic debridement of the soft tissue lesion may be all that is needed -LSB- 1 . In cases with an area of abraded articular cartilage on the anterolateral surface of the talar dome , further debridement of this region was required -LSB- 4 -RSB- . At long-term follow-up arthroscopic excision of soft tissue overgrowth and osteophytes proved to be an effective way of treating anterior impingement of the ankle in patients who had no narrowing of the joint space -LSB- 30 -RSB- . Patients with less than 2 years of ankle pain before surgery for anterior ankle impingement showed significant better scores in pain , swelling , ability to work , and engagement in sports -LSB- 31 -RSB- . The decision to perform an arthroscopy of the ankle is typically based on the patient 's history and physical examination -LSB- 9 , 19 , 28 -RSB- . Arthroscopically resection of the distal fascicle of the AITFL should be considered when there -LRB- Fig. 2 -RRB- : -LRB- 1 -RRB- is contact between the AITFL and the talus was prominent also in the beginning of plantarflexion-inversion of the ankle , -LRB- 2 -RRB- is increased contact between the talus and the ligament and this continued until maximum dorsiflexion with abrasion of the articular cartilage -LRB- the surgeon should remember that this finding may be normal -RRB- , -LRB- 3 -RRB- bending of the fascicle on the anterolateral edge of the talus with dorsiflexion and dorsiflexion-inversion , -LRB- 4 -RRB- is a distally inserting fascicle on the fibula , close to the origin of the ATFL on the fibula . This finding may be missed if the distraction is preserved throughout the procedure -LSB- 2 , 24 -RSB- . For this reason , temporarily relieving distraction is advised , when a distal fascicle is seen during arthroscopy -LSB- 2 , 22 -RSB- . Fig. 2There is contact between the distal fascicle of AITFL and the talus in dorsiflexion with bending of this fascicle Only three studies reported on the results of the treatment of AITFL impingement -LSB- 1 , 4 , 12 -RSB- . Akseki et al. -LSB- 1 -RSB- arthroscopically resected the distal fascicle of the AITFL in 21 patients with chronic ankle pain after an ankle sprain . During the procedure an impinging distal fascicle of the AITFL was found in all cases . Following anterolateral synovectomie , the fascicle was excised . At an average follow-up of 3 years good to excellent results were obtained in 17 patients . Nineteen patients were satisfied with the procedure and 17 patients returned to previous level of activity . Two patients who had mild laxity were graded as poor because neuromas of the terminal branches of the superficial peroneal nerve . These patients became asymptomatic after an injection of steroids . Bassett et al. -LSB- 4 -RSB- treated seven patients who had talar impingement by an AITFL and they were followed for a mean of 39 months . Two patients had arthroscopy and five had an arthrotomy . In all patients , a thickened distal fascicle of the AITFL was resected . Five patients also needed debridement of an area of abraded hyaline cartilage on the anterolateral aspect of the talar dome at the point where the distal fascicle made contact . Post-operative management consisted of 2 -- 4 days of immobilisation , followed by progressive weightbearing . Four patients reported no pain in their ankle or limitation in activity , and the results were considered to be excellent . Two patients reported improvement , with only occasional pain related to overuse , and the result was considered good . The last patient had been followed for only 6 months with excellent results . Horner and Liu -LSB- 12 -RSB- treated nine athletes with anterolateral ankle pain during and following athletic activities without history of ankle injury . After 3 months conservative management each patient was explored arthroscopically . Patients ' outcomes were rated subjectively and objectively based on the UCLA ankle scoring system . Pathological characteristics were easily identifiable through arthroscopy . The structure may be resected during this procedure without sacrificing the stability of the ankle joint . They report that good to excellent results can be expected from this procedure including in the highest level of athleticism at an average follow-up of 18 months . The decision to perform an arthroscopy of the ankle is typically based on the patient 's history and physical examination . When an impinging distal fascicle of the AITFL and an abraded anterolateral region of the talus were observed during an ankle arthroscopy , the surgeon should look for the criteria described above to decide whether it is pathological and needs to be resected . Conclusion Because of the lack of evidence only preliminary conclusions can be drawn . The AITFL started from the distal tibia , 5 mm in average above the articular surface , and descended obliquely between the adjacent margins of the tibia and fibula , anterior to the syndesmosis to the anterior aspect of the lateral malleolus . The incidence of the accessory fascicle differs very widely in the several studies . The presence of the distal fascicle of the AITFL and also the contact with the anterolateral talus is probably a normal finding . It may become pathological , however , due to anatomical variations and/or anterolateral instability of the ankle resulting from an ATFL injury . When observed during an ankle arthroscopy , the surgeon should look for the criteria described to decide whether it is pathological and considering resection of the distal fascicle .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O

ankle
impingement syndrome
anterior inferior tibiofibular ligament
accessory fascicle


The Importance of Preconception Care for Women With Disabilities Overall estimates of the number of women with disabilities in the United States range from 16.8 to 28.6 million , or approximately one in every five women -LSB- 1 -- 3 -RSB- . A large percentage of these women are in their reproductive years , and they often encounter greater obstacles to receiving health care than women without disabilities -LSB- 4 -RSB- . Some disabling conditions , such as spinal cord injury -LRB- SCI -RRB- and multiple sclerosis -LRB- MS -RRB- , have low prevalence rates but are more common in the reproductive age group . Such conditions are often associated with a greater severity of disability and increased need for specialized services , including prenatal care -LSB- 5 -RSB- . Health care professionals need to be aware of the reproductive health care issues facing women with disabilities and take every opportunity to address them . Sexuality and reproductive health issues have received inadequate attention for women with disabilities . Many women with disabilities desire children and are capable of conceiving , but face considerable pressure not to reproduce -LSB- 6 , 7 -RSB- . Although a growing number of women with disabilities are becoming pregnant , anecdotal evidence suggests that many of these women encounter negative attitudes towards their pregnancies and report difficulty receiving comprehensive prenatal care -LSB- 6 -RSB- . Women with disabilities who become pregnant face similar issues and concerns as pregnant women without disabilities . Although many pregnant women experience problems with weight gain , fatigue , fluid retention , bladder dysfunction , and urinary tract infections , these problems may be more serious in women with disabilities -LSB- 8 -RSB- . All women are concerned about the health of their unborn child . Like other women , women with disabilities express concern about the possibility of giving birth to a child with disabilities and may seek genetic counseling . On the other hand , not all disabled women embrace the idea of genetic testing . To these women the idea of genetic counseling raises psychosocial issues that may be difficult for them to consider -LSB- 6 -RSB- . Some disabling conditions pose unique problems during or after pregnancy . Complications associated with specific conditions , such as systemic lupus erythematosus -LRB- SLE -RRB- , SCI , or MS , can affect pregnancy and should be evaluated prior to conception -LSB- 8 -- 12 -RSB- . For example , an increased risk of fetal loss , growth retardation , premature placental aging with thrombosis , and preeclampsia are described in women with lupus -LSB- 9 -RSB- . Given the seriousness of these complications , ongoing monitoring and interventions to reduce them are critical . Pregnant women with SCI above the level of T6 also require special attention -LSB- 7 , 8 , 10 , 11 -RSB- . Such women have an increased risk of autonomic dysreflexia during all stages of pregnancy and , if it is not recognized and treated , autonomic dysreflexia can be fatal -LSB- 8 , 11 -RSB- . Although women with stable MS can safely carry a pregnancy to term , postpartum exacerbations of MS are quite common and occur in up to 30 % of women within one month of delivery -LSB- 11 , 12 -RSB- . Pregnancy management in women with disabilities should therefore include preconception counseling and evaluation to identify these and other potential problems that can occur throughout the course of the pregnancy . Health care professionals need to know that physical barriers , such as the lack of accessible scales or examination tables , present enormous and recurring obstacles to obtaining adequate prenatal care . Also , communication barriers , particularly for those who are deaf or hard of hearing , affect a women 's ability to receive appropriate medical care . In addition , there are some aspects of gynecologic health care such as informed consent , sedation , and contraceptive issues that are unique for women with developmental disabilities . A recent publication from the American College of Obstetricians and Gynecologists highlights ways to improve care for this group of women -LSB- 7 -RSB- . Preconception care is associated with improved pregnancy outcomes . It is recommended that primary care providers assess all women of reproductive age for their preconception risk conditions and provide or refer individuals for interventions as appropriate -LSB- 13 -RSB- . When caring for a woman with a disability , a team approach involving the primary physician , obstetrician , anesthesiologist , neurologist , physiatrist , and other allied health professionals such as occupational and physical therapists , is recommended -LSB- 6 -- 8 , 11 -RSB- . This is particularly important , as many obstetricians are unfamiliar with disability-related complications . A multidisciplinary approach , which involves nursing and social work , can further improve pregnancy outcomes . This shared approach to pregnancy management can also increase the level of comfort for women with disabilities , by knowing that more than one health care provider is looking out for their well-being . Preconception counseling for women with disabilities should address the medical , psychological , and social impact of a pregnancy -LSB- 10 -RSB- . During preconception counseling , it is particularly important to evaluate the prenatal and postpartum social support systems available to women with disabilities . Body changes such as decreased mobility or bladder dysfunction increase the need for personal assistance with routine activities of daily living . Many women with disabilities also benefit from an occupational therapy evaluation to assess environmental barriers and explore adaptive equipment and techniques that facilitate breast feeding and infant care . Health care providers might want to recommend that women with disabilities explore creative parenting strategies such as those offered by Through the Looking Glass , a national resource center on parenting with a disability , located in Berkeley , California -LSB- 6 , 14 -RSB- . An increasing number of women with disabilities are becoming pregnant . Despite this little is known about the reproductive experiences of these women . Population-based data describing the reproductive experiences of women with disabilities is virtually non-existent . Currently , disability is not regularly included in national or statewide surveys and surveillance tools related to pregnancy . Consequently , the true magnitude of health concerns , including reproductive issues , facing women with disabilities is largely unknown . To improve our understanding of these issues , disability should be included as a demographic variable in all national and state-level surveys addressing reproductive health issues . All women of childbearing age , including women with disabilities , should have access to preconception care that addresses the medical , psychological and social impacts of pregnancy . Women with disabilities can and do have healthy pregnancies . Accurate knowledge of disabling conditions and a shared multidisciplinary approach to pregnancy management are critical components for good pregnancy outcomes among women with disabilities .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O


Reticulon 4a/NogoA locates to regions of high membrane curvature and may have a role in nuclear envelope growth Reticulon 4a -LRB- Rtn4a -RRB- is a membrane protein that shapes tubules of the endoplasmic reticulum -LRB- ER -RRB- . The ER is attached to the nuclear envelope -LRB- NE -RRB- during interphase and has a role in post mitotic/meiotic NE reassembly . We speculated that Rtn4a has a role in NE dynamics . Using immuno-electron microscopy we found that Rtn4a is located at junctions between membranes in the cytoplasm , and between cytoplasmic membranes and the outer nuclear membrane in growing Xenopus oocyte nuclei . We found that during NE assembly in Xenopus egg extracts , Rtn4a localises to the edges of membranes that are flattening onto the chromatin . These results demonstrate that Rtn4a locates to regions of high membrane curvature in the ER and the assembling NE . Previously it was shown that incubation of egg extracts with antibodies against Rtn4a caused ER to form into large vesicles instead of tubules . To test whether Rtn4a contributes to NE assembly , we added the same Rtn4a antibody to nuclear assembly reactions . Chromatin was enclosed by membranes containing nuclear pore complexes , but nuclei did not grow . Instead large sacs of ER membranes attached to , but did not integrate into the NE . It is possible therefore that Rtn4a may have a role in NE assembly . 1 Introduction The nuclear envelope -LRB- NE -RRB- encloses the nucleus -LRB- Hetzer et al. , 2005 ; Goldberg , 2004 -RRB- and consists of two parallel sheets of membrane connected at nuclear pore complexes -LRB- NPCs -RRB- . The outer membrane is continuous with the rough endoplasmic reticulum -LRB- ER -RRB- . During mitosis and meiosis in higher eukaryotes the NE is dismantled . The lamina and NPCs are solubilised and the membranes disperse into the cytoplasm by retraction into the ER -LRB- Ellenberg et al. , 1997 ; Yang et al. , 1997 -RRB- or by vesiculation -LRB- Vigers and Lohka , 1991 -RRB- . During telophase the NE is reassembled around chromosomes in a multistage process , involving accumulation of different membrane populations and NE and NPC proteins . Evidence from Xenopus egg extracts suggest that there are least two vesicle populations -LRB- Vigers and Lohka , 1991 ; Macaulay and Forbes , 1996 ; Drummond et al. , 1999 -RRB- which fuse to form tubules and sheets -LRB- Wiese et al. , 1997 ; Goldberg et al. , 1992 ; Hetzer et al. , 2001 -RRB- which enclose the chromatin . Fusion of nuclear membranes requires hydrolysis of GTP -LRB- Macaulay and Forbes , 1996 -RRB- by Ran -LRB- Hetzer et al. , 2000 ; Zhang and Clarke , 2000 ; Zhang et al. , 2002 -RRB- and also the p97 -- UFD1 -- NPL4 complex -LRB- Hetzer et al. , 2001 -RRB- . The mechanisms of targeting and fusion are unknown . NPCs are apparent after a few minutes in egg extracts -LRB- Goldberg et al. , 1992 -RRB- , but NPC proteins accumulate in a temporal order in culture cells -LRB- Bodoor et al. , 1999 -RRB- as do structural intermediates in the NPC assembly process -LRB- Goldberg et al. , 1997 ; Kiseleva et al. , 2001 -RRB- . During S-phase the NE grows as the DNA content in the nucleus is increased -LRB- Winey et al. , 1997 -RRB- . The NE also grows during oogenesis . Although interphase NE growth is often considered separately from telophase , there are common features . Conceptually , once the chromatin is enclosed in telophase , there is no obvious difference between the subsequent growth phase and interphase growth . Like enclosure , growth requires the AAA-ATPase p97 , but instead of UFD1 and NPL4 it is complexed with p47 -LRB- Hetzer et al. , 2001 -RRB- , suggesting the mechanisms are related but distinct . Rtn4a/NogoA -LRB- hereafter referred to as Rtn4a -RRB- is a member of reticulon family and is one of three splice variants of the RTN4 gene -LRB- Oertle and Schwab , 2003 -RRB- . It has attracted much interest recently because of its inhibitory role in neurite outgrowth -LRB- Prinjha et al. , 2000 ; Yan et al. , 2006 -RRB- . Rtn4a has also been shown to localise to the ER -LRB- van de Velde et al. , 1994 -RRB- but is restricted to the tubular network and excluded from the peripheral ER sheets and NE -LRB- Voeltz et al. , 2006 -RRB- . Rtn4a was shown to be required for formation of ER tubules from sheets and vesicles -LRB- Voeltz et al. , 2006 -RRB- and it was suggested that reticulons could induce and stabilise the high curvature of the membrane required to maintain tubules . It is possible that the unusual topography of the reticulons , with their long putative transmembrane domains , could induce curvature when clustered . Rtn4a does not appear to be concentrated at the NE during interphase -LRB- Voeltz et al. , 2006 -RRB- possibly because the NE membrane bilayers consist of flat sheets with low membrane curvature . However , nuclear assembly involves considerable reorganisation of membrane topology . It is thought that ER tubules , shaped by Rtn4a -LRB- Voeltz et al. , 2006 -RRB- , feed into the NE -LRB- Ellenberg et al. , 1997 -RRB- and vesicles may also contribute -LRB- Vigers and Lohka , 1991 ; Liu et al. , 2003 ; Prunuske et al. , 2005 -RRB- . These vesicles and tubules have to be converted to a large flattened double sheet during NE assembly . Recently , it was shown that Rtn4a may have an essential role in NE disassembly in C. elegans -LRB- Audhya et al. , 2007 -RRB- . Therefore , we decided to test if Rtn4a could have a role in NE formation , both during interphase and telophase . We used a high resolution surface imaging technique , field emission in-lens scanning electron microscopy -LRB- feiSEM1 -RRB- to look at the structure of interphase growing NEs during Xenopus oogenesis and in telophase in vitro . We show that highly curved membrane regions of the forming or growing NEs preferentially contain Rtn4a . Such regions include the junctions between apparently fusing vesicles and the edges of flattening membranes . An antibody against Rtn4a was also shown to affect NE assembly . We therefore suggest that Rtn4a could have a role in NE formation and growth . 2 Results The ER connects to the NE in interphase and has been implicated in the post mitotic assembly of the NE -LRB- Ellenberg et al. , 1997 ; Yang et al. , 1997 ; Mattaj , 2004 -RRB- . Much of the interphase ER forms a tubular network which appears to feed into the NE during reassembly -LRB- Ellenberg et al. , 1997 ; Hetzer et al. , 2001 -RRB- . Rtn4a has been found to be involved in shaping the ER into tubules in specific regions -LRB- Voeltz et al. , 2006 -RRB- . Therefore , we speculated that if the ER needs to be in a tubular form to contribute to NE growth and assembly then Rtn4a may have a role . First we asked whether Rtn4a is associated with membranes that are contributing to NE growth , and then we investigated whether Rtn4a might be required for NE assembly . 2.1 Rtn4a is located at inter-membrane junctions between cytoplasmic vesicles near the NE During oogenesis in Xenopus , oocytes are arrested in pre-prophase when the nucleus grows to over 100 μM diameter . The NE must expand by the addition of membranes and assembly of lamina and NPCs . We previously observed , both in thin section TEM of whole oocytes and isolated nuclei and in feiSEM of isolated NEs , that growing stage III oocytes have more extraneous ER-like membranes associated with the NE than mature stage VI -LRB- Morozova and Kiseleva , 2006 -RRB- . FeiSEM analysis of the extraneous membranes localised near the NE showed that many were present as structures that look like long lines of inter-connected vesicles -LRB- Fig. 1a -RRB- . We are not certain of the origin of these structures but their surface has an ER-like appearance , with ribosome-like particles on the surface -LRB- Fig. 1b and d , arrowheads -RRB- . The vesicle-like structures are joined together by a short thin tubular connection of ∼ 20 nm diameter -LRB- Fig. 1b , white arrows -RRB- . The same structures were also observed in TEM thin sections of whole oocytes , showing that they are not artifacts of NE isolation or feiSEM specimen preparation -LRB- Fig. 1c -RRB- . We have also used different fixation methods -LRB- see Section 4.1 -RRB- . In TEM sections -LRB- Fig. 1c -RRB- the 20 nm diameter inter-connecting tubes are continuous with the vesicle membranes and therefore appear to be membrane bridges between vesicle-like structures . Anti-Rtn4a immuno-gold labelling with a previously characterised affinity purified anti Xenopus Rtn4a antibody -LRB- Voeltz et al. , 2006 -RRB- showed that Rtn4a was present on the surface of vesicles -LRB- Fig. 1f -RRB- . In the inter-connected vesicle structures , Rtn4a labelling was concentrated at the junctions between connected membrane structures where the membrane bridge is located -LRB- Fig. 1d and e -RRB- . Inter-connected larger membrane structures were also observed -LRB- Fig. 1g and h -RRB- where Rtn4a was located at the junction between them . Immuno-gold labelling of TEM sections of whole oocytes also shows localisation of Rtn4a at the contact point of adjacent vesicles . -LRB- Fig. 1i -RRB- . Rtn4a therefore appears to locate to the inter-connections between membrane structures -LRB- Fig. 1j -RRB- . These results show that Rtn4a locates to specific regions on NE associated cytoplasmic membranes . 2.2 Rtn4a is present on membranes attached to growing NEs We isolated NEs from stage III oocytes and immuno-gold labelled them for Rtn4a . There was some labelling of the ONM -LRB- Fig. 2a -RRB- but membrane structures associated with the NE were heavily labelled and in some cases , in a specific pattern . Fig. 2a is an image of the surface of a stage III NE showing NPCs , rough -LRB- ribosome-containing -RRB- vesicles -LRB- RV -RRB- , smooth -LRB- ribosome-free -RRB- vesicles -LRB- SV -RRB- , and rough membranes that appear to be flattening onto the ONM -LRB- FM -RRB- . We see that there is labelling of the ONM and vesicles are labelled to a varying degree . Smooth vesicles are not labelled , whereas rough ER type vesicles are . This shows that Rtn4a is associated with some but not all membranes that are associated with the ONM of a growing NE . 2.3 Rtn4a localises to the junction between the ONM and membranes attached to it Membrane structures could be seen attached to the ONM . Rtn4a appeared to be located near the point of contact between the membrane structures and the ONM -LRB- Fig. 2b -- d -RRB- . Therefore , it appears that Rtn4a accumulates both at junctions between ER-like membranes and between NE associated membranes that are attached to the ONM . NEs labelled with an antibody -LRB- CEL5C -RRB- to the ER protein ribophorin -LRB- Drummond et al. , 1999 -RRB- showed a more even distribution over flattened membranes , some vesicles and the outer nuclear membrane -LRB- Fig. 2e -RRB- . 2.4 Rtn4a localises to the edges of flattened membranes Some of the observed NE associated membranes had the appearance of collapsed spheres -LRB- Fig. 3a -RRB- whereas many are flattened -LRB- Fig. 3b -- f -RRB- . It is not always clear from feiSEM images whether such membranes are fused to the ONM or simply lying on top , so thin section TEM was carried out and showed continuity between the ONM and overlying flattened membrane structures -LRB- Fig. 4 , black arrow -RRB- . Points of contact between the ONM and cytoplasmic membrane structures were also observed -LRB- Fig. 4 , white arrows -RRB- . Potentially corresponding images were seen by feiSEM , in which the flattened membrane structures appear continuous with the NPC-containing ONM -LRB- Fig. 3b , arrows -RRB- . Using immuno-gold labelling , we observed that Rtn4a appeared to locate preferentially around the edges of the flattened membranes -LRB- Fig. 3d -- f -RRB- . This was quantified by counting the number of gold labels that were within 30 nm of the edge compared to greater than 30 nm from the edge -LRB- Fig. 5 -RRB- , which gave a ratio of approximately 2:1 , for edge compared to the interior . This was in contrast to the ER protein , ribophorin , which was distributed more away from the edge -LRB- Fig. 5 -RRB- . The preferred edge location suggests that Rtn4a tends to locate or accumulate at regions with the highest curvature . We conclude that Rtn4a marks the highly curved edges of flattened sheets of presumed ER membrane attached to the ONM . 2.5 Rtn4a is present at the edges of membranes that are flattening onto the chromatin During telophase , membranes also flatten onto chromatin -LRB- Goldberg et al. , 1992 ; Macaulay and Forbes , 1996 -RRB- . We wanted to test if the edge location of Rtn4a that we observed in growing oocyte NEs also occurred in chromatin bound flattening membranes . To do this , we used a cell free system from Xenopus eggs which can be used to assemble nuclei in vitro -LRB- Goldberg and Allen , 1993 ; Lohka and Masui , 1984 -RRB- . Nuclei were isolated from assembly reactions , fixed and immuno-gold labelled for Rtn4a . At early stages -LRB- 2 -- 5 min -RRB- vesicles bind to the chromatin and flatten -LRB- Goldberg et al. , 1992 ; Wiese et al. , 1997 -RRB- . In such vesicles , Rtn4a localises preferentially around the edges -LRB- Fig. 6a and b -RRB- , compared to a general ER protein , ribophorin -LRB- antibody CEL5C -- Drummond et al. , 1999 -RRB- , which was more randomly distributed -LRB- Fig. 6d -RRB- . To show this formally we counted the distribution of gold particles located within 30 nm of the vesicle edge , or further away in 30 nm increments , using images from three separate experiments -LRB- Fig. 6e -RRB- . This shows that Rtn4a , compared to ribophorin , is preferentially located near the membrane edge . Therefore , as in growing oocytes , Rtn4a preferentially locates to the edges of flattening membranes . At later stages of assembly when the chromatin is enclosed , and there are no longer any highly curved membrane edges -LRB- except at the nuclear pores -RRB- , the Rtn4a distribution on the NE appeared random and low level -LRB- Fig. 6c -RRB- . This is consistent with Rtn4a 's preference for curved membranes -LRB- Voeltz et al. , 2006 -RRB- . Although there are highly curved membranes in the nuclear pores we see no Rtn4a labelling there . The high membrane curvature at the NPC might be maintained by nucleoporins which could exclude the accumulation of Rtn4a , or it is possible that Rtn4a is present but not detected by the antibody due to epitope masking . 2.6 Disrupting Rtn4a prevents NE growth Although Rtn4a is not preferentially located to the NE during interphase -LRB- Voeltz et al. , 2006 -RRB- , our results in stage III oocytes show that it does locate to membranes that are attached to the interphase NE and suggested the possibility that it may be involved in NE assembly or growth . Therefore , we wanted to investigate if Rtn4a might be required for NE assembly . We incubated egg extracts with a previously characterised anti Xenopus Rtn4a antibody directed against the cytoplasmic facing N-terminus -LRB- Voeltz et al. , 2006 -RRB- which is specific to Rtn4a and not present in other Rtn4 spliced variants or other reticulon proteins . This antibody inhibits the formation of ER tubules in similar egg extracts -LRB- Voeltz et al. , 2006 -RRB- and likewise we found that it inhibited ER tubule formation . Instead , ER membranes assembled into large vesicular structures -LRB- Fig. 7b -RRB- rather than tubules as seen in the no-antibody control reactions -LRB- Fig. 7a -RRB- . These large membrane structures are ER-derived because they have ribosomes on their surface . This shows that the anti-Rtn4a antibody had a dominant effect on the formation of ER tubules . To determine if the Rtn4a antibody affected NE assembly , the same egg extracts were incubated with the antibody -LRB- see Section 4.1 -RRB- or with buffer or with an irrelevant antibody , before adding sperm chromatin to initiate nuclear assembly . Controls showed rapid binding , fusion and flattening of vesicles onto chromatin , following by chromatin decondensation , enclosure and NE growth to form large roughly spherical nuclei > 10 microns in diameter , as expected -LRB- Goldberg et al. , 1997 ; Wiese et al. , 1997 and Fig. 7c -RRB- . In the presence of the Rtn4a antibody we found that nuclei did assemble with an apparently completely enclosed unbroken NE -LRB- Fig. 7d -- g -RRB- that had apparently normal NPCs -LRB- Fig. 7i , black arrows -RRB- . However , the chromatin failed to decondense and remained as a dense sperm-shaped object surrounded by NE -LRB- Fig. 7d -- g -RRB- . To quantify this apparent NE growth defect , we measured the two-dimensional area occupied by each nucleus -LRB- excluding attached membranes , see below -RRB- as viewed from above by feiSEM -LRB- Fig. 7h -RRB- . Nuclei in Rtn4a antibody-inhibited reactions had ∼ 80 % reduced area compared to controls , confirming the growth defect . There were in addition , large -LRB- several microns -RRB- membrane structures extending from the nucleus -LRB- Fig. 7d -- g , arrows -RRB- . The extensions are clearly rough ER-like and contain ribosomes -LRB- Fig. 7i , arrowheads -RRB- . There is a sharp demarcation between the chromatin attached membrane which contains NPCs and the membrane extensions which contain ribosomes but not NPCs -LRB- Fig. 7i , white arrows -RRB- . The extensions are clearly continuous with the NPC-containing NE . Therefore , it appears that perturbing Rtn4a does not prevent enclosure of the chromatin by NE . However , the chromatin and NE do not expand despite the attachment of large ER-like sacs to the ONM . These sacs are similar to the large ER vesicle formed in the cytosol in the presence of the antibody , except they are attached to the NE . Antibodies and reagents against other NE and ER proteins do not have such an affect on nuclear assembly or ER tubulation . Addition of wheat germ agglutinin , which binds to certain nucleoporins -LRB- Hanover et al. , 1987 -RRB- , to extracts results in nuclei without NPCs but it has no effect on ER tubulation and does not result in membrane extensions -LRB- Goldberg et al. , 1997 -RRB- . Addition of anti-nucleoporin antibodies -LRB- Mab414 and QE5 -RRB- -LRB- unpublished results -RRB- or depletion of extracts with antibodies to specific nucleoporins , Nup214 -LRB- Walther et al. , 2002 -RRB- or Nup153 -LRB- Walther et al. , 2001 -RRB- , also affected NPC structure but again does not affect the ER or NE membranes . Depletion of lamin B3 resulted in small spherical nuclei with normal NPCs but had no obvious effect on ER tubules or membrane extensions -LRB- Goldberg et al. , 1995 -RRB- . The CEL5C antibody against the ER protein ribophorin -LRB- Drummond et al. , 1999 -RRB- also did not affect tubulation or membrane extensions when added to extracts -LRB- unpublished result -RRB- . Similar experiments were also done -LRB- Voeltz et al. , 2006 -RRB- using antibodies to IP3R and TRAPα which also did not affect ER tubule formation . Therefore , we believe that the growth defects and extensions are a specific effect of the anti-Rtn4a antibody used in this study . 3 Discussion We have found that Rtn4a is localised to junctions between membrane structures and at the edges of flattened membranes associated with growing NEs both in oocytes and in nuclei assembling in vitro . This is the first direct evidence to support the proposal that Rtn4a locates to regions of high curvature -LRB- Voeltz et al. , 2006 -RRB- . It is a unique localisation that has not been seen for other NE or ER proteins . We believe this localisation may be driven by Rtn4a itself rather than by interacting with other proteins because high level over-expression of Rtn4a in COS cells -LRB- Voeltz et al. , 2006 -RRB- and the related Rtn1 in yeast -LRB- De Craene et al. , 2006 -RRB- does not change the proteins ' localisations and therefore is unlikely to rely on other titratable factors . The location of Rtn4a at the junction between cytoplasmic membranes and NE membranes suggested to us the possibility that Rtn4a may also have a role in NE assembly . Addition of the Rtn4a antibody to nuclear assembly reactions allowed NE formation in egg extracts but chromatin remained condensed and the NE did not expand . Therefore , the antibody has a dominant effect on the growth phase of NE assembly but not on the initial enclosure and NPC assembly . As Rtn4a is an integral membrane protein that appears to be involved in shaping membranes -LRB- Voeltz et al. , 2006 -RRB- we suggest that the effect of the antibody is to perturb nuclear membrane dynamics and assembly , as shown previously for the ER -LRB- Voeltz et al. , 2006 -RRB- . One possible speculation for the localisation of Rtn4a at membrane -- membrane junctions in oocytes is that it may take part in fusion or stabilisation of membrane curvature during fusion . Inter-membrane fusion involves transitory extremes of curvature involving membrane stalks between vesicles -LRB- Smeijers et al. , 2006 ; Yang and Huang , 2002 -RRB- . Rtn4a could be involved in stalk formation or stabilisation in certain NE membranes . We do not know if these membranes in oocytes are actively fusing membranes or if they are more stable or intermediate structures . Rtn4a could be involved in stabilising the conformation of these junctions by maintaining the high membrane curvature to facilitate membrane flow into the NE . Our in vitro antibody inhibition experiments suggest that ER-like membranes can attach to the NE when Rtn4a is perturbed but the NE fails to grow . When Rtn4a is perturbed the ER membranes are converted to large sacs , which can attach to the NE but fail to contribute to NE expansion . This suggests the possibility that the organisation of the interface between the NE and ER , as observed in oocytes , may be important for NE growth . Therefore we speculate that a possible function of localising Rtn4a to the inter-membrane junctions between ONM and ER is to maintain a particular interfacial organisation which contributes to the movement of membrane into the NE . Our results argue against a model in which membranes simply diffuse into the NE during assembly and growth -LRB- Ellenberg et al. , 1997 -RRB- because the NE fails to grow when Rtn4a is perturbed despite the attachment of ER membranes to the NE . Rtn4a is not only located at the inter-membrane junctions but also throughout the tubular ER , where it appears to be involved in maintaining the tubularity -LRB- Voeltz et al. 2006 ; Fig. 7 -RRB- . Therefore , the tubular nature of the ER may be essential for its function in providing membrane for NE growth , at least in egg extracts . The Rtn4a antibody does not inhibit the initial formation of NE around chromatin . This NE appears normal : it is flattened , fused and contains NPCs . Because it contains NPCs we can conclude that the contributing membranes carried the integral membrane nucleoporins such as POM121 and gp210 enabling NPC formation . This suggests that Rtn4a may only be required during the growth phase and that membranes that contribute to initial enclosure and NPC formation -LRB- Yang et al. , 1997 ; Drummond et al. , 1999 -RRB- are not perturbed by the antibody . We have also found that Rtn4a locates specifically to the edges of flattening membranes in growing and assembling NE in both in vivo and in vitro experiments . As Rtn4a has been implicated in the formation of highly curved membrane regions , it is possible to speculate that Rtn4a is important for formation of flattened sheets , by stabilisation of the high curvature at the edge regions . However , this may be a non essential function in the initial stages of NE assembly , which occur in the presence of the antibody . 4 Conclusions We have shown for the first time that Rtn4a partitions , within a single membrane structure , to the region of highest curvature , supporting the model that reticulons preferentially locate to curved membranes -LRB- Voeltz et al. , 2006 -RRB- and have a function related to membrane curvature . We have also observed that it locates to membrane junctions and other highly curved regions of membranes that may be involved in NE growth . Concordantly , an Rtn4a antibody perturbs NE growth . We therefore hypothesise that Rtn4a may have a role in maintaining functional ER -- NE junctions during NE growth and/or the ER must be tubular to contribute to NE growth . 4.1 Materials and methods 4.1.1 Isolation and fixation Xenopus oocyte for feiSEM Stage III and VI NEs were isolated in 5:1 buffer , spread , fixed in 2 % glutaraldehyde , 0.2 % tannic acid , 10 mM Tris -- HCl -LRB- pH 7.4 -RRB- and 0.3 mM MgCl2 and processed for feiSEM as described previously -LRB- Kiseleva et al. 2004 -RRB- . In some experiments tannic acid , which helps preserve protein filament -LRB- Maupin and Pollard , 1983 -RRB- was omitted , as it can effect membrane preservation -LRB- MWG unpublished -RRB- , but the same membrane structures were observed . Samples were coated with chromium using a Cressington 308R with additional cryo-pump or an Edwards Auto306 with cryo-pump to a nominal thickness of 2 nm . They were viewed using a Hitachi S-5200 feiSEM at 10 kV accelerating voltage . 4.1.2 Fixation Xenopus oocyte for TEM Oocytes were fixed -LRB- 2.5 % glutaraldehyde , 0,1 M Hepes -RRB- for 1 h , then washed twice in 0.1 M Hepes buffer and postfixed 1 h in 1 % OsO4 in ddH2O at 4 °C , stained 2 h in 1 % aqueous uranyl acetate , washed in water , dehydrated through ethanol series and embedded in Agar-100 -LRB- Agar Scientific , UK -RRB- . Sections were stained with Lead citrate and viewed with Leo 910 -LRB- Germany -RRB- TEM at 80 kV . 4.1.3 Immuno-TEM Stage III oocytes were fixed in 4 % formaldehyde -LRB- TAAB Labs -RRB- in Ringers -LRB- 111 mM Nacl , 1.9 mM KCl , 1.1 mM CaCl2 , 2.4 mM NaHCO3 , pH 7.0 -RRB- overnight at 4 °C , washed three times in Ringers , stain in 2 % uranyl acetate in Ringers for 2 h at 4 °C , then washed in water twice , dehydrated in 30 % ethanol , then further dehydrated by freeze substitution as follows through a series of ethanol -LRB- 30 % at 4 °C for 1 h , 50 % at − 20 °C for 1 h , 70 % at − 20 °C for 1 h , 95 % at − 20 °C for 2 h -RRB- , embedded to LR Gold -LRB- Agar -RRB- at − 20 °C and polymerised in gelatin capsules under UV light at − 15 °C for 48 h. Ultrathin sections were cut and attached to nickel grids then incubated with 1 % BSA in PBS for 30 min , washed in PBS , incubated with 1:100 dilution anti-Rtn4a rabbit antibody for 1 h , washed in PBS and incubated with a goat anti-rabbit secondary antibody conjugated to 10 nm colloidal gold -LRB- Amersham -RRB- for 1 h and washed with PBS . Sections were stained with lead citrate for 2 min . 4.1.4 Immuno-gold labelling and feiSEM imaging Affinity purified polyclonal antibody -LRB- 40 M stock -RRB- against N-terminal domain of Xenopus Rtn4a -LRB- Voeltz et al. , 2006 -RRB- was diluted 1:100 with PBS . Nuclei were isolated and fixed for 20 min in 3.7 % formaldehyde , 5:1 buffer , washed three times with PBS , incubated in PBS , 1 % BSA 30 min , washed in PBS , and incubated 1 -- 3 h primary antibody , PBS . Samples were washed three times in PBS , and incubated 1 h with 10 nm gold-conjugated secondary goat anti-rabbit antibody -LRB- Amersham Corp. -RRB- . As negative controls , we used gold-conjugated secondary antibody diluted 1:100 in PBS . All samples were then washed three times in PBS then in 10 mM Tris -- HCl and processed for feiSEM as above . Both secondary electron images -LRB- for structure -RRB- and backscatter electron images -LRB- for gold label position -RRB- were collected simultaneously . Using Adobe Photoshop , the backscatter image was superimposed onto the secondary image as a separate layer . The positions of the gold particles were marked with a yellow dot and then the backscatter image removed . 4.1.5 In vitro nuclear assembly Egg extracts were prepared as described previously -LRB- Goldberg et al. , 1997 -RRB- . To perturb Rtn4a , extract was incubated with 4 μM affinity purified Xenopus Rtn4a antibody -LRB- Voeltz et al. , 2006 -RRB- on ice for 20 min then 1000 sperm chromatin per microlitre was added and the extract warmed to room temperature for 2 -- 60 min for assembly . In controls an equal volume of either buffer or irrelevant antibody was added , which had no detectable effect on nuclear assembly . Extracts were resuspended in Membrane Wash Buffer -LRB- MWB : 250 mM sucrose , 50 mM KCl , 2.5 mM MgCl2 , 10 mM Hepes , pH 7.4 -RRB- and centrifuged at 1000g at 4 °C for 10 min onto silicon chips -LRB- Agar Scientific Ltd -RRB- and immersed in Membrane Fix -LRB- 150 mM sucrose , 1 mM MgCl2 , 80 mM Pipes -- KOH , pH 6.8 , 2 % paraformaldehyde , 0.25 % glutaraldehyde -RRB- for 10 min and processed for feiSEM as previously described -LRB- Goldberg et al. , 1997 -RRB- . Samples were imaged at 3 kV accelerating voltage . 4.1.6 Antibody labelling of in vitro nuclei Extract -LRB- 5 μl -RRB- was resuspended in MWB and centrifuged at 1000g at 4 °C for 10 min onto silicon chips -LRB- Agar Scientific , UK -RRB- . Chips were immersed in Membrane Fix without glutaraldehyde for 10 min , washed in PBS , immersed in PBS +100 mM glycine 10 min , washed in PBS , immersed in 1 % fish skin gelatine -LRB- Sigma -RRB- for 1 h , then 1 μM primary antibody for 1 h , washed 3 times 5 min in PBS , incubated with 1:50 dilution anti-rabbit secondary antibody conjugated to 10 nm gold -LRB- Amersham -RRB- for 1 h , washed 3 times 5 min and 1 times 15 min in PBS and fixed in Membrane fix with 1 % glutaraldehyde . Samples were then processed for feiSEM as previously described -LRB- Goldberg et al. , 1997 -RRB- .

O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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