RESUMO
Biological studies have relied on two complementary microscope technologies - light (fluorescence) microscopy and electron microscopy. Light microscopy is used to study phenomena at a global scale to look for unique or rare events, and it also provides an opportunity for live imaging, whereas the forte of electron microscopy is the high resolution. Traditionally light and electron microscopy observations are carried out in different populations of cells/tissues and a 'correlative' inference is drawn. The advent of true correlative light-electron microscopy has allowed high-resolution imaging by electron microscopy of the same structure observed by light microscopy, and in advanced cases by video microscopy. Thus a rare event captured by low-resolution imaging of a population or transient events captured by live imaging can now also be studied at high resolution by electron microscopy. Here, the potential and difficulties of this approach, along with the most impressive breakthroughs obtained by these methods, are discussed.
Assuntos
Microscopia Eletrônica/métodos , Microscopia/métodos , Microscopia de Vídeo/métodosRESUMO
Protein kinase D (PKD) is a cytosolic protein, which upon binding to the trans-Golgi network (TGN) regulates the fission of transport carriers specifically destined to the cell surface. We have found that the first cysteine-rich domain (C1a), but not the second cysteine-rich domain (C1b), is sufficient for the binding of PKD to the TGN. Proline 155 in C1a is necessary for the recruitment of intact PKD to the TGN. Whereas C1a is sufficient to target a reporter protein to the TGN, mutation of serines 744/748 to alanines in the activation loop of intact PKD inhibits its localization to the TGN. Moreover, anti-phospho-PKD antibody, which recognizes only the activated form of PKD, recognizes the TGN-bound PKD. Thus, activation of intact PKD is important for binding to the TGN.
Assuntos
Cisteína/metabolismo , Proteína Quinase C/metabolismo , Rede trans-Golgi/metabolismo , Substituição de Aminoácidos , Anticorpos/farmacologia , Linhagem Celular , Ativação Enzimática/fisiologia , Genes Reporter , Glutationa Transferase/genética , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica/fisiologia , Proteína Quinase C/antagonistas & inibidores , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae. Based on this evidence, we have proposed that PC-I is transported across the Golgi stacks by the cisternal maturation process. However, most secretory cargoes are small, freely diffusing proteins, thus raising the issue whether they move by a transport mechanism different than that used by PC-I. To address this question we have developed procedures to compare the transport of a small protein, the G protein of the vesicular stomatitis virus (VSVG), with that of the much larger PC-I aggregates in the same cell. Transport was followed using a combination of video and EM, providing high resolution in time and space. Our results reveal that PC-I aggregates and VSVG move synchronously through the Golgi at indistinguishable rapid rates. Additionally, not only PC-I aggregates (as confirmed by ultrarapid cryofixation), but also VSVG, can traverse the stack without leaving the cisternal lumen and without entering Golgi vesicles in functionally relevant amounts. Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.
Assuntos
Fibroblastos/metabolismo , Complexo de Golgi/metabolismo , Glicoproteínas de Membrana , Transporte Proteico , Fenômenos Fisiológicos da Pele , Animais , Anticorpos , Linhagem Celular , Fibroblastos/ultraestrutura , Congelamento , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde , Humanos , Processamento de Imagem Assistida por Computador , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Eletrônica , Microscopia Imunoeletrônica , Coelhos , Proteínas Recombinantes/metabolismo , Pele/metabolismo , Pele/ultraestrutura , Proteínas do Envelope Viral/metabolismoRESUMO
Integrating the pleomorphic membranes of the intermediate compartment (IC) into the array of Golgi cisternae is a crucial step in membrane transport, but it is poorly understood. To gain insight into this step, we investigated the dynamics by which cis-Golgi matrix proteins such as GM130 and GRASP65 associate with, and incorporate, incoming IC elements. We found that GM130 and GRASP65 cycle via membranous tubules between the Golgi complex and a constellation of mobile structures that we call late IC stations. These stations are intermediate between the IC and the cis-Golgi in terms of composition, and they receive cargo from earlier IC elements and deliver it to the Golgi complex. Late IC elements are transient in nature and sensitive to fixatives; they are seen in only a fraction of fixed cells, whereas they are always visible in living cells. Finally, late IC stations undergo homotypic fusion and establish tubular connections between themselves and the Golgi. Overall, these features indicate that late IC stations mediate the transition between IC elements and the cis-Golgi face.