Реорганизация актинового цитоскелета, лежащая в основе движения клеток
Наши исследования реорганизации актиновой сети при движении фибробластов подтвердили существование двух зон на ведущем крае клетки, отличающихся по строению и динамическим характеристикам — ламеллиподии и ламеллы. Мы впервые выделили и разделили молекулярные механизмы, регулирующие динамику актина в этих зонах, и показали, что быстрый ретроградный ток актина в ламеллиподии определяется Агр 2/3… Читать ещё >
Содержание
- Список сокращений
- Актуальность проблемы
- Цели и задачи
- Научная новизна и практическая ценность
- ЛИТЕРАТУРНЫЙ ОБЗОР. 17 L ОРГАНИЗАЦИЯ АКТИНОВОГО ЦИТОСКЕЛЕТА КЛЕТОК ПОЗВОНОЧНЫХ
- ГЛАВА 1. БЕЛКОВАЯ ОРГАНИЗАЦИЯ АКТИНОВОГО ЦИТОСКЕЛЕТА
- 1. 1. Актин — основной белок микрофиламснтов
- 1. 2. Белки, индуцирующие полимеризацию актина
- 1. 2. 1. Arp2/3 (Actin-related Protein) комплекс
- 1. 2. 2. Формипы
- 1. 2. 3. УУН2-домен-содержащие актиновые нуклеаторы
- 1. 3. Белки, регулирующие динамику актиновых филаментов в клетке
- 1. 3. 1. Кэпирующие белки
- 1. 3. 2. VASP (Vasodilator-stimulatedphosphoprotein)
- 1. 3. 3. ADF/кофилин (Actin Depolimerasing Factor)
- 1. 4. Белки, регулирующие трехмерную организацию актина
- ГЛАВА 2. РАСПРЕДЕЛЕНИЕ АКТИНА В ПОДВИЖНОЙ КЛЕТКЕ ФИБРОБЛАСТЕ)
- 2. 1. Строение актинового цитоскелета фибробласта
- 2. 2. Дискуссия о строении актииовой сети в ламеллиподии — ветвятся 29 ли актиновые филаменты в живых клетках?
- ГЛАВА 3. СТРОЕНИЕ И ФУНКЦИИ ФОКАЛЬНЫХ АДГЕЗИЙ, РАЗНЫЕ 33 ТИПЫ АДГЕЗИЙ, СВЯЗЬ С ЦИТОСКЕЛЕТОМ
- ГЛАВА 4. РЕГУЛЯЦИЯ КЛЕТОЧНОГО ДВИЖЕНИЯ МАЛЫМИ ГТФ-азами СЕМЕЙСТВА RHO
- ОСТАЮЩИЕСЯ ВОПРОСЫ ш
- ДВИЖЕНИЕ ТРАНСФОРМИРОВАННЫХ КЛЕТОК
- ГЛАВА 5. СПОСОБЫ МИГРАЦИИ ТРАНСФОРМИРОВАННЫХ КЛЕТОК
- 5. 1. Коллективная миграция
- 5. 2. Мезенхимальиый способ миграции
- 5. 3. Амебоидный способ миграции
- 5. 4. Пластичность, как свойство опухолевых клеток
- ГЛАВА 6. ОСОБЕННОСТИ ЦИТОСКЕЛЕТА ТРАНСФОРМИРОВАННЫХ КЛЕТОК
- ГЛАВА 7. ИЗМЕНЕНИЕ СТРУКТУРЫ И СТРОЕНИЯ АДГЕЗИОННЫХ КОНТАКТОВ КЛЕТКА-МАТРИКС В ПРОЦЕССЕ КАНЦЕРОГЕНЕЗА
- 7. 1. Изменение белкового состава фокальных адгезий в процессе канцерогенеза
- 7. 2. Приобретение нового типа адгезионных структур в процессе канцерогенеза. Подосомы
- 7. 3. Дальнейшая дедифференцировка ведет к ослаблению взаимодействия клеток с ВКМ
- 7. 4. Изменение состава внеклеточного матрикса в процессе канцерогенеза
- ГЛАВА 8. УЧАСТИЕ МАЛЫХ ГТФаз В ПРОЦЕССАХ ИНВАЗИИ И
- КАНЦЕРОГЕНЕЗА ОСТАЮЩИЕСЯ ВОПРОСЫ МАТЕРИАЛЫ И МЕТОДЫ Клеточные культуры
- Методы световой микроскопии
- Фазовый контраст с компьютерным усилением
- Флуоресцентная микроскопия
- Конфокальная микроскопия
- Интерференционно-отражательная микроскопия (ШРмикроскопия)
- TIRF- м икроскоп ия
- Видеосъемка
- Трансфекция живых клеток, использованные конструкты 55 Анализ динамики формирования и подвижности клеточных структур. Кимограммы
- Использованные ингибиторы и модуляторы движения
- Анализ морфологии клеток
- Электронная микроскопия
- Метод платиновых реплик 58 Коррелятивная электронная микроскопия, негативный контраст
- Электронная томография
- Исследование клеточной подвижности 60 Подвижность индивидуальных клеток. Анализ характера псевдоподиальной активности
- Анализ характера и направления миграции
- Миграция одиночных клеток в редкой культуре
- Анализ миграции клеток в экспериментальную рану
- Анализ миграции и инвазии клеток в камерах Бойдена
- Инвазия в матригель
- Определение активности Ras
- Зимография
- Статистическая обработка результатов
- РЕЗУЛЬТАТЫ
- I. ИССЛЕДОВАНИЕ РЕОРГАНИЗАЦИИ ЦИТОСКЕЛЕТА, ЛЕЖАЩЕЙ В
- ОСНОВЕ ДВИЖЕНИЯ НОРМАЛЬНЫХ КЛЕТОК
- ГЛАВА 1. ИНИЦИАЛЬНЫЕ ПРОЦЕССЫ ДВИЖЕНИЯ КЛЕТОК
- 1. 1. ПЕРЕСТРОЙКИ ЦИТОСКЕЛЕТА ЭПИТЕЛИОЦИТОВ ПРИ ИНИЦИАЦИИ ДВИЖЕНИЯ ПОД ДЕЙСТВИЕМ СКЭТТЕР- 66 ФАКТОРА (HGF/SF)
- 1. 2. РАЗДЕЛЕНИЕ НА ФРАГМЕНТЫ МНОГОЯДЕРНЫХ ЭПИТЕЛИАЛЬНЫХ КЛЕТОК MDCK ПОД ДЕЙСТВИЕМ 68 HGF/SF
- 1. 3. Исследование активации движения клеток под действием форболового эфира РМА
- ГЛАВА 2. ИССЛЕДОВАНИЕ ВКЛАДА ДВУХ СОСТАВЛЯЮЩИХ -ПОЛИМЕРИЗАЦИИ АКТИНА И АКТО-МИОЗИНОВОЙ СОКРАТИМОСТИ В ПОДВИЖНОСТЬ КЛЕТОК
- 2. 1. Исследование влияния ингибиторов сократимости и полимеризации актина на распластывание фибробластов
- 2. 1. 1. Динамика распластывания контрольных фибробластов
- 2. 1. 2. Влияние ингибиторов сократительной активности миозина 81 II (У27 632 и блеббистатина на динамику распластывания нормальных фибробластов)
- 2. 1. 3. Влияние ингибиторов полимеризации актина латрункулина, А и цитохалазина Д на динамику распластывания нормальных фибробластов
- 2. 2. Исследование влияния ингибиторов сократимости и полимеризации актина на способность клеток к миграции
- 2. 2. 1. Движение нормальных фибробластов в рану
- 2. 2. 2. Движение клеток в присутствии ингибиторов полимеризации актина
- 2. 2. 3. Движение клеток в присутствии ингибиторов сократимости
- 2. 2. 4. Формирование монослоя нарушается в присутствии ингибиторов миозиновой сократимости
- 2. 1. Исследование влияния ингибиторов сократимости и полимеризации актина на распластывание фибробластов
- 3. 1. Две зоны ведущего края клетки — ламеллиподия и ламелла
- 3. 2. Разные молекулярные механизмы, регулирующие движение актина в ламелле и ламеллиподии
- 3. 3. Роль формирования первичных фокальных контактов в динамической организации ведущего края клетки
- 3. 3. 1. Формирование первичных фокальных контактов локально блокирует ламеллиподиальный ток актина
- 3. 3. 2. Ингибирование ламеллиподиального быстрого ретроградного тока блокирует формирование инициальных контактов
- 4. 1. Модели для исследования динамики ФА
- 4. 2. Формирование VASP-положительных ФА
- 4. 3. Динамическая колоколизация VASP и винкулина и ультраструктура винкулин-положительных инициальных контактов
- 4. 4. Динамическая колокализация VASP и зиксина и ультраструктура зиксин-положительных инициальных контактов
- 4. 5. Динамическая колокализация VASP и бета-З-интегрина
- 4. 6. Исследование образования инициальных ФА у фибробластов ЗТЗ с помощью электронной томографии
- II. ИССЛЕДОВАНИЕ ИЗМЕНЕНИЙ, ВОЗНИКАЮЩИХ В РЕЗУЛЬТАТЕ ТРАНСФОРМАЦИИ
МОРФОЛОГИЯ, СТРОЕНИЕ АКТИНОВОГО ЦИТОСКЕЛЕТА И АССОЦИИРОВАННЫХ С НИМ СТРУКТУР И АНАЛИЗ КРАЕВОЙ АКТИВНОСТИ У КОНТРОЛЬНЫХ И RAS-ТРАНСФОРМИРОВАННЫХ ФИБРОБЛАСТОВ Морфология контрольных и Ras-трансформированных фибробластов
Исследование динамики ведущего активного края контрольных и Ras-трансформированных фибробластов
МОРФОЛОГИЯ, СТРОЕНИЕ АКТИНОВОГО ЦИТОСКЕЛЕТА И АССОЦИИРОВАННЫХ С НИМ СТРУКТУР И АНАЛИЗ КРАЕВОЙ АКТИВНОСТИ У КОНТРОЛЬНЫХ И SV40-ТРАНСФОРМИРОВАННЫХ ФИБРОБЛАСТОВ Морфология и цнтоскелет контрольных и SV40трансформированыых фибробластов
Распределение и характер псевдоподиальной активности контрольных и 8У40-трансформированыых фибробластов
7.2.1. Морфометрические измерения периметра клеток
7.2.2. Распределение различных форм активности вдоль клеточного края
7.2.3. Распределение маркера образования протрузий Агр2/3-комплекса по периметру клетки
7.3. Ультраструктура актинового цитоскелета у контрольных и SV40-трансформироваиых фибробластов
7.4. Исследование динамики ведущего активного края контрольных и 177 8У40-трансформированных фибробластов
7.4.1. Частота протрузий и раффлов
ГЛАВА 8. МОРФОЛОГИЯ, СТРОЕНИЕ АКТИНОВОГО ЦИТОСКЕЛЕТА И АССОЦИИРОВАННЫХ С НИМ СТРУКТУР- АНАЛИЗ КРАЕВОЙ АКТИВНОСТИ У ПОДКОЖНЫХ ФИБРОБЛАСТОВ ЧЕЛОВЕКА ПО СРАВНЕНИЮ С КЛЕТКАМИ ФИБРОСАРКОМЫ
8.1. Морфологическое описание исследуемых клеточных культур
8.2. Строение актинового цитоскелета и ассоциированных с ним 181 структур у контрольных подкожных фибробластов и клеток фибросаркомы НТ
8.3. Распределение и характер пссвдоподиальной активности
8.4. Исследование динамики ведущего активного края контрольных фибробластов и клеток фибросаркомы НТ
ГЛАВА 9. ЛОКОМОТОРНОЕ ПОВЕДЕНИЕ НОРМАЛЬНЫХ И
ТРАНСФОРМИРОВАННЫХ ФИБРОБЛАСТОВ В КУЛЬТУРЕ.
9.1. Изменение локомоторного поведения в системе Ras-трансформации.
9.2. Изменение локомоторного поведения в системе SV-40 трансформации.
9.3. Анализ локомоторного поведения контрольных подкожных фибробластов и клеток фибросаркомы НТ
9.4. Оценка инвазивных способностей трансформированных фибробластов
ГЛАВА 10. ИССЛЕДОВАНИЕ ВОЗМОЖНЫХ МОЛЕКУЛЯРНЫХ МЕХАНИЗМОВ, ЛЕЖАЩИХ В ОСНОВЕ ПРИОБРЕТЕНИЯ КЛЕТКАМИ ИНВАЗИВНОГО ФЕНОТИПА.
10.1. Анализ активности матриксных металлопротеаз в исследуемых линиях.
10.2. Анализ вклада малой ГТФ-азы КЬо в формирование «трансформированного» фенотипа.
10.3. Выяснение роли уровня активных кислородных радикалов в формировании «трансформированного» фенотипа
ОБСУЖДЕНИЕ
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