Bernard Hoflack

Regulation of membrane traffic and bone remodeling

Previous and current research

Our group has been interested in understanding basic processes of lysosome biogenesis, an essential process for homeostasis of eukaryotic cells. We focus on 1) the mechanisms by which soluble and membrane proteins as well as lipids are sorted from the secretory pathway for subsequent transport to the endosomal/lysosomal system, 2) how these processes are regulated, especially during cell differentiation as observed with osteoclasts, which acquire the property of building-up an extracellular lysosome in order to digest bone. This prompted us to become interested in the biology of bone remodeling.

Much of our progress came from proteomic screens performed on in vitro reconstitution systems recapitulating key steps of lysosome biogenesis, in particular the selective interaction of the AP-1 and AP-3 coats with membranes. We have now identified the two sorting machineries involved, composed each of ≈50 different proteins that belong not only to sorting devices like coat components, but also to devices involved in actin polymerization and membrane fusion. This highlights the complexity of protein-protein interactions required for coordinating protein sorting and transport. Our progress also came from proteomic and genomic screens performed on osteoclasts and their precursors. These screens allowed us to identify a large number of proteins, in particular effectors of small GTPases, that are good candidates for establishing the cell polarity of osteoclasts and for remodeling their membrane traffic and actin dynamics, three essential processes required for efficient bone degradation. The role of interesting candidates is now investigated using RNA interference and appropriate functional assays monitoring cell polarity, membrane traffic or actin dymanics in mature osteoclasts.

Future prospects and goals

Capitalizing on our progress, our research follows three major directions in membrane and bone biology.

1. One focus of our group is to gain a comprehensive understanding of lysosomes biogenesis. We rely on the combination of biological and biophysical techniques applied to in vitro and in vivo systems to understand basic aspects of membrane traffic, in particular how coat assembly, actin polymerization and membrane fusion are coordinated. Second, we rely on high-throughput RNA interference-based functional screens to identify new components and to understand how lysosome biogenesis is regulated.

2. Another focus is the cell biology of osteoclasts. Whereas it is critical to obtain a large comprehensive view of signaling cascades regulating osteoclast differentiation, a process that we can now investigate using RNA interference-based functional screens, it is also important to have a deeper molecular understanding of how osteoclasts function in bone degradation. Of particular interest is actin dynamics (podosome formation), a process regulated by the Src kinase, and membrane/actin cytoskeleton interactions. During bone degradation, several intracellular compartments involved in secretion of hydrolytic enzymes required for bone digestion or uptake and transcytosis of digested bone material are concentrated around the podosome/actin-rich sealing zone of osteoclasts.

3. An important aspect in bone remodeling, i.e. cycles of destruction and rebuilding occurring continuously throughout live, is the coordinated action of the bone digesting osteoclasts and the bone rebuilding osteoblasts. It is now clear that signaling molecules expressed by osteoblasts control osteoclastogenesis. Using co-culture systems, we have accumulated evidence that osteoclasts could control some aspects of osteoblast-mediated bone rebuilding. We now want to investigate further these interesting possibilities using appropriate mouse model systems.

About

Selected publications

Baust T. Czupalla C. Krause E. Bourel-Bonnet L. Hoflack B. (2006): Proteomic analysis of AP-1A coats selectively assembled on liposomes. Proc. Natl. Acad. Sci. USA 103: 3159-3164.

Czupalla C, Mansukoski H, Riedl T, Thiel D, Krause E, Hoflack B. (2006): Proteomic Analysis of Lysosomal Acid Hydrolases Secreted by Osteoclasts: Implications for Lytic Enzyme Transport and Bone Metabolism. Mol Cell Proteomics 1:134-143.

Czupalla C, Mansukoski H, Pursche T, Krause E, Hoflack B. (2005): Comparative study of protein and mRNA expression during osteoclastogenesis. Proteomics. 15:3868-75.

Waguri S, Dewitte F, Le Borgne R, Rouille Y, Uchiyama Y, Dubremetz JF, Hoflack B. (2003): Visualization of TGN to endosome trafficking through fluorescently labeled MPR and AP-1 in living cells. Mol Biol Cell. 1:142-55.

Rohn WM, Rouille Y, Waguri S, Hoflack B. (2000): Bi-directional trafficking between the trans-Golgi network and the endosomal/lysosomal system (review). J. Cell Sci., 113: 2093-2101.

Rouille Y, Rohn W, Hoflack B. (2000): Targeting of lysosomal proteins (review). Semin. Cell Dev. Biol., 11: 165-171.

Le Borgne R, Hoflack B. (1998): Mechanisms of protein sorting and coat assembly: insights from the clathrin-coated vesicle pathway (review). Curr. Opin. Cell Biol., 10: 499-503.

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