Correlative microscopy: a potent tool for the study of rare or unique cellular and tissue events.

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.

Recruitment of protein kinase D to the trans-Golgi network via the first cysteine-rich domain.

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.

Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae.

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.