Phosphoinositide Regulation of Integrin Trafficking Required for Muscle Attachment and Maintenance

Muscles must maintain cell compartmentalization when remodeled during development and use. How spatially restricted adhesions are regulated with muscle remodeling is largely unexplored. We show that the myotubularin (mtm) phosphoinositide phosphatase is required for integrin-mediated myofiber attachments in Drosophila melanogaster, and that mtm-depleted myofibers exhibit hallmarks of human XLMTM myopathy. Depletion of mtm leads to increased integrin turnover at the sarcolemma and an accumulation of integrin with PI(3)P on endosomal-related membrane inclusions, indicating a role for Mtm phosphatase activity in endocytic trafficking. The depletion of Class II, but not Class III, PI3-kinase rescued mtm-dependent defects, identifying an important pathway that regulates integrin recycling. Importantly, similar integrin localization defects found in human XLMTM myofibers signify conserved MTM1 function in muscle membrane trafficking. Our results indicate that regulation of distinct phosphoinositide pools plays a central role in maintaining cell compartmentalization and attachments during muscle remodeling, and they suggest involvement of Class II PI3-kinase in MTM-related disease

Who Needs Microtubules? Myogenic Reorganization of MTOC, Golgi Complex and ER Exit Sites Persists Despite Lack of Normal Microtubule Tracks

A wave of structural reorganization involving centrosomes, microtubules, Golgi complex and ER exit sites takes place early during skeletal muscle differentiation and completely remodels the secretory pathway. The mechanism of these changes and their functional implications are still poorly understood, in large part because all changes occur seemingly simultaneously. In an effort to uncouple the reorganizations, we have used taxol, nocodazole, and the specific GSK3-β inhibitor DW12, to disrupt the dynamic microtubule network of differentiating cultures of the mouse skeletal muscle cell line C2. Despite strong effects on microtubules, cell shape and cell fusion, none of the treatments prevented early differentiation. Redistribution of centrosomal proteins, conditional on differentiation, was in fact increased by taxol and nocodazole and normal in DW12. Redistributions of Golgi complex and ER exit sites were incomplete but remained tightly linked under all circumstances, and conditional on centrosomal reorganization. We were therefore able to uncouple microtubule reorganization from the other events and to determine that centrosomal proteins lead the reorganization hierarchy. In addition, we have gained new insight into structural and functional aspects of the reorganization of microtubule nucleation during myogenesis

Special features of vesicle trafficking in skeletal muscle cells

Abstract Skeletal muscles are composed of long, multinucleated cells called myofibers, which are highly differentiated cells and therefore unique in structure. In the present study the organization of the endocytic and exocytic pathways in isolated rat skeletal myofibers was defined with confocal and electron microscopic methods. In isolated myofibers the I band areas were shown to be active in endocytosis. The sorting endosomes were distributed in a cross-striated fashion while the recycling and late endosomal compartments were located to perinuclear areas and interfibrillar spaces, where they followed the course of microtubules. Protein trafficking in the different stages of muscle cell differentation was also analyzed. The studies with L6 myoblasts and myotubes showed that during myogenesis varying fractions of different viral glycoproteins were sorted from the endoplasmic reticulum (ER) into a specific compartment that did not recycle with the Golgi apparatus. This compartment is suggested to be the sarcoplasmic reticulum (SR). The studies with living muscle cells showed further changes in vesicle trafficking taking place during myogenesis. With GFP-tagged tsO45G protein, transport containers were detected in 20% of the infected myofibers, while all infected L6 myoblasts or myotubes showed intense movement of corresponding structures. We also detected significant differences between the pre-and post-Golgi traffickings in myofibers. When the distribution of the ER in adult myofibers was studied, the confocal microscopic data showed that the labeling patterns of the rough endoplasmic reticulum (RER) and the SR markers were different. Blocking of different cargo proteins in the RER revealed two discrete distribution patterns, neither of them identical with the SR. The collected electron microscopic data supported the idea that in mature myofibers there are two separate RER compartments. We suggest that the RER compartment capable of export function located around the myonuclei and on the Z lines, while the non-exporting RER compartment localized to terminal cisternae and probably took care of the synthesis of the SR proteins

Cross-border knowledge transfer and innovation in the European neighbourhood: Tourism cooperation at the Finnish-Russian border

Knowledge transfer and innovation cooperation between the EU and its neighbours has remained weakly developed. To promote this cooperation, the EU has set up initiatives for the European neighbourhood. The issue has, however, received very limited scholarly attention in the field of tourism. This research gap is addressed here via interview data collected from participants in tourism related EU-funded projects in the Finnish-Russian cross-border region. These underline the importance of EU-funding in facilitating knowledge transfer and innovation between Finland and Russia. While language issues, and differences in business culture and administrative/legislative systems between the two countries, constitute barriers for practical cross-border cooperation, it is cross-border differences in culture and technological capabilities that drive cross-border knowledge transfer and innovation in the cross-border region. The paper concludes with policy recommendations for promoting future cross-border cooperation in innovation and tourism

Tieto- ja viestintätekniikan pedagoginen vaikuttavuus pohjoisessa Suomessa

Abstract The purpose of this study was to investigate the pedagogical impact of information and communication technology (ICT) from the perspectives of the teacher, student and school. The study involved both a qualitative and a quantitative component. The qualitative data consisted of teacher (N = 33) interviews, questionnaires directed to teachers (N = 32) and student (N = 90) interviews in twelve northern Finnish primary and lower secondary schools. The aim of the qualitative study was to study what kind of pedagogical teaching practices the teachers were using to support learning, what kind of learning skills and information society skills the educational use of ICT has inspired in the students, and what the structures and dimensions of the teachers' networks are like. The quantitative data consisted of a questionnaire addressed to 6th-graders (N = 1868) and 9th-graders (N = 4132). The sample was national in coverage. The quantitative research focused on students' learning processes, especially their motivation and cognitive strategies and the impact of ICT on these processes. It was also studied how the students' own interests and the school's role were related to their learning strategies and beliefs about learning. The qualitative research results indicated that teachers used ICT mostly to support students' individual and independent learning. Teachers utilized ICT quite little as a tool for knowledge building and knowledge sharing (such as problem-based or collaborative learning). The findings also indicated that the teachers used ICT to enrich their existing practices. The students considered tasks requiring information retrieval, evaluation of information and teamwork skills to be the most challenging and at the same time most inspiring. The results indicated that assignments based on students' individual work did not inspire them to learn. Further, the impact of ICT was significant on teachers who participated in different networks. Active networks inspired teachers to plan how to use ICT in their teaching and also added to collaboration and sharing of expertise between the networked participants. The interaction in the teachers' networks focused on technical issues, however, and collaboration related to the pedagogical use of ICT was rare. The quantitative research results indicated that the students' enthusiasm for using ICT and their experiences of ICT being supported by the school were related to their attitudes towards studying and learning new concepts, their learning motivation and their own ideas of their abilities as learners. In conclusion, we can say that teachers should have stronger views on appropriate approaches to the pedagogical use of ICT in teaching. The goals and objectives for the use of ICT should become the common cause of the entire school community. To put pedagogical reforms into effect, the schools should pay increased attention to evaluate and develop the pedagogical aims of ICT, to establish a school culture that supports the educational use of ICT, and to promote the networking of teachers and sharing of information with the various actors and stakeholders.Tiivistelmä Tämän tutkimuksen tavoitteena oli tarkastella tieto- ja viestintätekniikan pedagogista vaikuttavuutta opettajan, oppilaan ja koulun näkökulmasta. Tutkimus muodostui laadullisesta ja määrällisestä tutkimusosuudesta. Laadullinen tutkimusaineisto koostui 12 eri pohjoisen Suomen alakoulun ja yläkoulun opettajien (N = 33) haastattelusta, opettajille suunnatuista kyselylomakkeista (N = 32) ja oppilaiden (N = 90) haastattelusta. Laadullisen tutkimuksen tarkoituksena oli selvittää millaisia ovat opettajien käyttämät oppimista tukevat pedagogiset opetuskäytännöt, millaisia oppimisen taitoja ja tietoyhteiskuntataitoja tieto- ja viestintätekniikan opetuskäyttö on oppilaissa saanut aikaan ja millaisia ovat opettajaverkostojen rakenteet ja ulottuvuudet. Määrällinen tutkimusaineisto koostui kyselytutkimuksesta ja siihen vastasi yhteensä 6000 6-luokkalaista (N = 1868) ja 9-luokkalaista (N = 4132) oppilasta. Otos oli valtakunnallinen. Määrällisessä tutkimuksessa selvitettiin, millainen vaikutus tieto- ja viestintätekniikan käytöllä on oppilaan oppimisstrategioihin, erityisesti kognitiivisiin ja motivationaalisiin tekijöihin. Tutkimuksessa selvitettiin myös oppilaiden oman kiinnostuksen ja koulun roolin vaikutusta oppilaiden oppimisstrategioihin ja oppimista kuvaaviin uskomuksiin. Laadullisen tutkimuksen tulokset osoittivat, että opettajat käyttivät eniten oppilaiden yksilöllistä ja itsenäistä oppimista tukevia opetuskäytäntöjä. Opettajat hyödynsivät tieto- ja viestintätekniikkaa ymmärtävän oppimisen, kuten tutkivan ja ongelmakeskeisen oppimisen tai yhteisöllisen oppimisen, tukena varsin vähän. Tulokset osoittivat myös, että opettajat nivoivat tieto- ja viestintätekniikan käytön olemassa oleviin pedagogisiin käytäntöihin. Oppilastutkimuksen tulokset osoittivat, että oppilaat kokivat haastavimmaksi ja samalla myös innostavimmaksi tehtävät, jotka edellyttivät tiedon hakua, tiedon arviointia ja ryhmätyötaitoja. Oppilastutkimuksen tulokset viittaavat siihen, että yksilöllistä oppimista tukevat tehtävät eivät innostaneet oppilaita oppimaan. Tutkimustulokset opettajaverkostoista osoittivat, että tieto- ja viestintätekniikan vaikuttavuus oli merkittävää erilaisiin verkostoihin osallistuneisiin opettajiin. Toimivat verkostot lisäsivät opettajien tieto- ja viestintätekniikan opetuskäytön suunnittelua sekä verkoston toimijoiden välistä yhteistyötä ja asiantuntijuuden jakamista. Opettajaverkostojen vuorovaikutus keskittyi kuitenkin lähinnä tieto- ja viestintätekniikan teknisen käytön kysymyksiin ja tieto- ja viestintätekniikan pedagogiseen opetuskäyttöön liittyvä yhteistyö oli harvinaisempaa. Määrällisen tutkimuksen tulokset osoittivat, että oppilaan innostus sekä hänen kokemansa koulun tuki tieto- ja viestintätekniikkaan olivat yhteydessä hänen suhtautumiseensa opiskeluun ja uuden oppimiseen, opiskelumotivaatioon ja käsityksiin omista kyvyistään oppijana. Johtopäätöksenä voidaan todeta, että opettajilla tulisi olla vahvempi käsitys tarkoituksenmukaisista tieto- ja viestintätekniikan pedagogisista opetuskäytön ratkaisuista. Tieto- ja viestintätekniikan opetuskäytön tavoitteet ja päämäärät olisi nostettava koko kouluyhteisön asiaksi. Pedagogisten uudistusten aikaansaamiseksi koulujen olisi kiinnitettävä huomiota tieto- ja viestintätekniikan pedagogisten tavoitteiden arviointiin ja kehittämiseen, tieto- ja viestintätekniikan opetuskäyttöä tukevan toimintakulttuurin luomiseen sekä edistettävä opettajien verkostoitumista ja tiedon jakamista eri tahojen ja toimijoiden kanssa

Endocytosis in skeletal muscle fibers

Defining the organization of endocytic pathway in multinucleated skeletal myofibers is crucial to understand the routing of membrane proteins, such as receptors and glucose transporters, through this system. Here we analyzed the organization of the endocytic trafficking pathways in isolated rat myofibers. We found that sarcolemmal-coated pits and transferrin receptors were concentrated in the I band areas. Fluid phase markers were taken up into vesicles in the same areas along the whole length of the fibers and were then delivered into structures around and between the nuclei. These markers also accumulated beneath the neuromuscular and myotendinous junctions. The recycling compartment, labeled with transferrin, appeared as perinuclear and interfibrillar dots that partially colocalized with the GLUT4 compartment. Low-density lipoprotein, a marker of the lysosome-directed pathway, was transported into sparsely distributed perinuclear and interfibrillar dots that contacted microtubules. A majority of these dots did not colocalize with internalized transferrin, indicating that the recycling and the lysosome-directed pathways were distinct. In conclusion, the I band areas were active in endocytosis along the whole length of the multinucleated myofibers. The sorting endosomes distributed in a cross-striated fashion while the recycling and late endosomal compartments showed perinuclear and interfibrillar localizations and followed the course of microtubules. (C) 1999 Academic Press

3D bioprinting of the kidney:hype or hope?

Abstract Three-dimensional (3D) bioprinting is an evolving technique that is expected to revolutionize the field of regenerative medicine. Since the organ donation does not meet the demands for transplantable organs, it is important to think of another solution, which may and most likely will be provided by the technology of 3D bioprinting. However, even smaller parts of the printed renal tissue may be of help, e.g. in developing better drugs. Some simple tissues such as cartilage have been printed with success, but a lot of work is still required to successfully 3D bioprint complex organs such as the kidneys. However, few obstacles still persist such as the vascularization and the size of the printed organ. Nevertheless, many pieces of the puzzle are already available and it is just a matter of time to connect them together and 3D bioprint the kidneys. The 3D bioprinting technology provides the precision and fast speed required for generating organs. In this review, we describe the recent developments in the field of developmental biology concerning the kidneys; characterize the bioinks available for printing and suitable for kidney printing; present the existing printers and possible printing strategies. Moreover, we identify the most difficult challenges in printing of the kidneys and propose a solution, which may lead to successful bioprinting of the kidney