Бесплатный автореферат и диссертация по биологии на тему
Центры организации микротрубочек в интерфазных и митотических меланофорах шпорцевой лягушки in vivo
ВАК РФ 03.00.11, Эмбриология, гистология и цитология
Содержание диссертации, кандидата биологических наук, Рубина, Ксения Андреевна
ВВЕДЕНИЕ.
ОБЗОР ЛИТЕРАТУРЫ.
1. Структурно-функциональная характеристика центросомы.
2. Центросомный и ядерный циклы.
3. Организация митотического веретена.
4. Особенности ЦОМТ в дифференцированных клетках.
5. Нецентросомальные ЦОМТ.
6. Динамика микротрубочек.
7. Микротрубочки в дифференцированных клетках. Полярность клеток.
8. Регуляция ЦОМТ и микротрубочек в зависимости от внешнего сигнала.
9. Участие микротрубочек в транспорте внутриклеточных органелл.
10. Общая характеристика меланофоров.
11. Строение меланофоров.
МАТЕРИАЛЫ И МЕТОДЫ.
1. Иммунофлуоресцентная микроскопия и компьютерная обработка изображений.
2. Электронная микроскопия.
3. Обработка колцемидом.
4. Трехмерная реконструкция.
РЕЗУЛЬТАТЫ.
1. Иммунофлуоресцентный анализ интерфазных меланофоров.
2. Электронномикроскопический анализ интерфазных и митотических клеток.
1. Интерфазные клетки в состоянии дисперсии.
2. Интерфазные клетки в состоянии агрегации.
3. Митотические клетки.
3. Обработка колцемидом интерфазных меланофоров. Световая микроскопия.
4. Ультраструктура интерфазных меланофоров, обработанных колцемидом.
1. Меланофоры в состоянии агрегации.
2. Меланофоры в состоянии дисперсии.
3. Смена гормонов в присутствии колцемида. Агрегированные меланофоры.
4. Смена гормонов в присутствии колцемида. Дисперсные меланофоры.
5. Трехмерная реконструкция формы клеток.
ОБСУЖДЕНИЕ.
ВЫВОДЫ.
Введение Диссертация по биологии, на тему "Центры организации микротрубочек в интерфазных и митотических меланофорах шпорцевой лягушки in vivo"
Центросома была открыта более ста лет назад, но сегодня, в связи с разработкой новых биохимических, генетических методов в сочетании с электронной и световой микроскопией, она вызывает особый интерес, о чем свидетельствует тот факт, что международный симпозиум по клеточной биологии в 1997 году был полностью посвящен центросоме. До сих пор нет четких ответов на вопросы: Как происходит сборка и организация центросомы? Какова функция центриолей? Каков механизм нуклеации микротрубочек на центросоме? Каково происхождение центриолей и центросомы в эволюционном аспекте? Как центросома влияет на динамику микротрубочек и каков механизм этой регуляции?
Цитоскелет играет важную роль в процессах внутриклеточного транспорта и морфогенезе клеток, реализуя информацию линейного двумерного кода ДНК в трехмерную систему пространственных взаимодействий клеточных компонентов (Andersen, 1999). Микротрубочки являются одним из компонентов цитоскелета; центросома служит основным центром организации микротрубочек (ЦОМТ) в большинстве эукариотических клеток (Brinkley, 1985). Микротрубочки играют важную роль при делении клеток, поляризации и миграции, внутриклеточном транспорте и других процессах. Благодаря своей способности к дисперсии и агрегации пигментных гранул при индукции соответствующими гормонами, меланофоры стали излюбленным объектом при изучении процессов и механизмов внутриклеточного транспорта (Bikle et al., 1966; Schliwa and Bereiter-Hahn, 1973; Schliwa, 1975; Schliwa et al., 1978; McNiven et al., 1984; McNiven and Porter, 1986; Rozdzial and Haimo, 1986a,b; McNiven and Ward 1988; Thaler and Haimo, 1990; Blanchard et al., 1991; Fujii, 1993; Rodionov et al., 1991; Rodionov et al., 1994; Rodionov and Borisy, 1997; Rogers et al., 1997; Rodionov and Borisy, 1998;). Считается, что транспорт меланосом происходит вдоль микротрубочек при участии моторных белков, ассоциированных с микротрубочками (Rodionov et al. 1991; Rodionov and Borisy, 1994; Rogers et al. 1997).
Изменения структуры центросомы и цитоскелета при индукции дисперсии и агрегации описаны на интерфазных меланофорах рыб in vivo и in vitro (Bikle et al., 1966; Schliwa and Bereiter-Hahn, 1973; Schliwa 1978; Schliwa et al. 1978; Schliwa 1986; Rodionov et al., 1991). Имеются данные об организации цитоскелета в культивируемых меланофорах шпорцевой лягушки (Xenopus laevis) (Rollag and Adelman, 1993). Однако мало что известно об организации центросомы и сети микротрубочек в меланофорах Xenopus laevis in vivo.
Дермальные меланофоры головастика шпорцевой лягушки - клетки гигантского размера, со сложно устроенной цитоплазмой и множеством отростков, имеющих сложное пространственное распределение. Известно, что архитектура клеток в системе in vitro и in vivo может существенно различаться, а, следовательно, механизмы клеточного ответа и физиологические реакции клеток в культуре и в ткани тоже могут быть различными (Ingber, 1993). В связи этим одной из задач настоящей работы стал анализ структуры, числа и локализации ЦОМТ, а также системы микротрубочек и формы клеток интерфазных дермальных меланофорах шпорцевой лягушки в различных физиологических состояниях (дисперсии и агрегации) in vivo.
Имеющиеся данные литературы свидетельствуют о том, что меланофоры амфибий способны вступать в митоз (Pehlemann, 1967; Стародубов и Голиченков, 1979; Стародубов и Голиченков, 1979а; Стародубов и Голиченков, 1979b; Ide, 1982; Семерджиева и др., 1992). В отличие от интерфазных меланофоров, митотические меланофоры на стадии метафазы сохраняют отросчатую форму и не отвечают на гормональные стимулы, вызывающие дисперсию и агрегацию в интерфазных клетках (Стародубов и Голиченков, 1979а; Стародубов и Голиченков 1979b; Стародубов и Голиченков, 1988; Ide, 1982).
Подробного анализа особенностей ЦОМТ в митотических меланофорах до сих пор не проводилось. В связи с этим в настоящей работе была поставлена задача провести исследование структуры ЦОМТ, организации микротрубочек и клеточной формы митотических меланофоров Xenopus laevis in vivo.
В литературе имеются противоречивые данные о влиянии антитубулиновых агентов на число и организацию микротрубочек в хроматофорах, а также на возможность транспорта пигментных гранул в таких условиях. Известно, что в ряде клеток обработка колцемидом вызывает разборку микротрубочек, а последующая инкубация в среде без колцемида позволяет выявить число и локализацию ЦОМТ. В определенных условиях колцемид может вызывать инактивацию центросомы (De Brabander et al., 1981). Колцемид может служить хорошим инструментом при анализе числа ЦОМТ, стабильности микротрубочек, формы и функциональной активности центросомы и процессов внутриклеточного транспорта в двух различных физиологических состояниях меланофоров - при дисперсии и агрегации. В связи с этим в настоящей работе была поставлена задача провести анализ влияния разных доз колцемида на структуру и функцию центросомы, на стабильность сети микротрубочек в дисперсных и агрегированных интерфазных меланофорах Xenopus laevis in vivo. Также стояла задача провести светомикроскопическое исследование возможности дисперсии и агрегации меланосом при индукции этих процессов соответствующим гормоном в присутствии колцемида и проанализировать ультраструктуру этих меланофоров.
ОБЗОР ЛИТЕРАТУРЫ
Заключение Диссертация по теме "Эмбриология, гистология и цитология", Рубина, Ксения Андреевна
выводы
1. Центросома интерфазных и митотических меланофоров Xenopus имеет строение, в целом, характерное для центросом большинства соматических клеток in vivo и in vitro.
2. Особенностью центросомы меланофоров Xenopus являются: а), способность центросомы интерфазных клеток образовывать первичную ресничку; б), присутствие в интерфазных и митотических меланофорах множественных центриолей; в), возможность активации всех центриолей клетки в качестве ЦОМТ в интерфазных и митотических меланофорах.
3. Для митотических и интерфазных меланофорв в состоянии агрегации характерен трехзональный тип организации ЦОМТ: центриоли, окруженные ПЦМ; центросфера и астральная лучистость с радиальным расположением органелл вдоль микротрубочек.
4. Микротрубочки обнаруживаются в отростках интерфазных и митотических меланофоров, независимо от присутствия меланосом.
5. Микротрубочки в дисперсных меланофорах существенно стабильнее микротрубочек в агрегированных меланофорах. Самыми стабильными являются центросомные микротрубочки в дисперсных меланофорах.
6. Меланофоры являются отросчатыми клетками; переход клеток из агрегированного состояния в дисперсное в интерфазе и из интерфазы в митоз сопровождается изменением клеточной формы и организации ЦОМТ.
7. Структура центросомы интерфазных меланофоров чувствительна к воздействию возрастающих концентраций колцемида (10"3 М - 10"6 М). Повышение концентрации колцемида до 10° М вызывает коллапс центросомы в агрегированных меланофорах. Колцемид в концентрации 10'6 М не препятствует действию МСГ, который индуцирует дисперсию меланосом, при этом в некоторых клетках наблюдается инактивация центросомы в качестве ЦОМТ. Мелатонин же в присутствие колцемида вызывает аномальную агрегацию, при этом в ряде клеток наблюдается активация центросомы в качестве ЦОМТ.
8. Дополнительных ЦОМТ, отличных от центросомы, в интерфазных меланофорах не обнаружено ни в норме, ни после воздействия колцемидом. Это позволяет считать центросому единственным ЦОМТ.
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455. Ф фокусы схождения микротрубочек ПР - первичная ресничка X - хромосомы к - коллаген4
456. Рис. 1. Световая микроскопия. Перемещение пигмента в меланофорах при агрегации под действием мелатонина (А-Д) и при дисперсии под действием а-МСГ (Е-Ж). Масштабный отрезок: 100 ¡лт.
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Рубина, Ксения Андреевна
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кандидата биологических наук
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Москва, 1999
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ВАК 03.00.11
- Исследование функций белков, ассоциированных с микротрубочками, с помощью специфических антител
- Динамика развития пигментной системы личинок шпорцевой лягушки
- Цитофизиологические последствия ультрафиолетового микрооблучения центросомы в клетках культуры ткани
- Динамика физиологических реакций пигментной системы в онтогенезе бесхвостых амфибий
- Внецентросомные детерминанты организации микротрубочек в интерфазных клетках
