Tom Kirchhausen

T-cell engagement of dendritic cells rapidly rearranges MHC class II transport

Assembly of major histocompatibility complex (MHC) molecules, which present antigen in the form of short peptides to T lymphocytes, occurs in the endoplasmic reticulum; once assembled, these molecules travel from the endoplasmic reticulum to their final destination. MHC class II molecules follow a route that takes them by means of the endocytic pathway, where they acquire peptide, to the cell surface. The transport of MHC class II molecules in ‘professional’ antigen-presenting cells (APCs) is subject to tight control and responds to inflammatory stimuli such as lipopolysaccharide. To study class II transport in live APCs, we replaced the mouse MHC class II gene with a version that codes for a class II molecule tagged with enhanced green fluorescent protein (EGFP). The resulting mice are immunologically indistinguishable from wild type. In bone-marrow-derived dendritic cells, we observed class II molecules in late endocytic structures with transport patterns similar to those in Langerhans cells observed in situ. We show that tubular endosomes extend intracellularly and polarize towards the interacting T cell, but only when antigen-laden dendritic cells encounter T cells of the appropriate specificity. We propose that such tubulation serves to facilitate the ensuing T-cell response.

An emergency response team for membrane repair

On demand, rapid Ca2+-triggered homotypic and exocytic membrane-fusion events are required to repair a torn plasma membrane, and we propose that this emergency-based fusion differs fundamentally from other rapid, triggered fusion reactions. Emergency fusion might use a specialized protein and organelle emergency response team that can simultaneously promote impromptu homotypic fusion events between organelles and exocytic fusion events along the vertices between these fusion products and the plasma membrane.

Use of dynasore, the small molecule inhibitor of dynamin, in the regulation of endocytosis.

The large GTPase dynamin is essential for clathrin-dependent coated-vesicle formation. Dynasore is a cell-permeable small molecule that inhibits the GTPase activity of dynamin1, dynamin2 and Drp1, the mitochondrial dynamin. Dynasore was discovered in a screen of approximately 16,000 compounds for inhibitors of the dynamin2 GTPase. Dynasore is a noncompetitive inhibitor of dynamin GTPase activity and blocks dynamin-dependent endocytosis in cells, including neurons. It is fast acting (seconds) and its inhibitory effect in cells can be reversed by washout. Here we present a detailed synthesis protocol for dynasore, and describe a series of experiments used to analyze the inhibitory effects of dynasore on dynamin in vitro and to study the effects of dynasore on endocytosis in cells.

The First Five Seconds in the Life of a Clathrin-Coated Pit

Coated pits assemble by growth of a clathrin lattice, which is linked by adaptors to the underlying membrane. How does this process start? We used live-cell TIRF imaging with single-molecule EGFP sensitivity and high temporal resolution to detect arrival of the clathrin triskelions and AP2 adaptors that initiate coat assembly. Unbiased object identification and trajectory tracking, together with a statistical model, yield the arrival times and numbers of individual proteins, as well as experimentally confirmed estimates of the extent of substitution of endogenous by expressed, fluorescently tagged proteins. Pits initiate by coordinated arrival of clathrin and AP2, which is usually detected as two sequential steps, each of one triskelion with two adaptors. PI-4,5-P2 is essential for initiation. The accessory proteins FCHo1/2 are not; instead, they are required for sustained growth. This objective picture of coated pit initiation also shows that methods outlined here will be broadly useful for studies of dynamic assemblies in living cells.

Molecular Structure, Function, and Dynamics of Clathrin-Mediated Membrane Traffic

Clathrin is a molecular scaffold for vesicular uptake of cargo at the plasma membrane, where its assembly into cage-like lattices underlies the clathrin-coated pits of classical endocytosis. This review describes the structures of clathrin, major cargo adaptors, and other proteins that participate in forming a clathrin-coated pit, loading its contents, pinching off the membrane as a lattice-enclosed vesicle, and recycling the components. It integrates as much of the structural information as possible at the time of writing into a sketch of the principal steps in coated-pit and coated-vesicle formation.

IMAGING ENDOCYTIC CLATHRIN STRUCTURES IN LIVING CELLS

Our understanding of the clathrin-dependent endocytic pathway owes much to new visualization techniques. Budding coated pits and clathrin-coated structures are transient molecular machines with distinctive morphological characteristics, and fluorescently labeled versions of a variety of marker proteins have given us a tantalizing glimpse of the dynamics of the system in living cells. Recent live-cell imaging studies have revealed unexpected modes of coat assembly, with distinct kinetics, distinct recruitment of associated proteins, distinct requirements for the participation of actin and its accessory proteins, and apparently distinct mechanisms of membrane deformation. A crucial issue is to connect the events detected by light microscopy with the structures and properties of the molecular constituents. Here, I outline descriptions of coat assembly in different circumstances that are consistent with what is known from X-ray crystallography and electron microscopy.

Cisternal Organization of the Endoplasmic Reticulum during Mitosis

The endoplasmic reticulum (ER) of animal cells is a single, dynamic, and continuous membrane network of interconnected cisternae and tubules spread out throughout the cytosol in direct contact with the nuclear envelope. During mitosis, the nuclear envelope undergoes a major rearrangement, as it rapidly partitions its membrane-bound contents into the ER. It is therefore of great interest to determine whether any major transformation in the architecture of the ER also occurs during cell division. We present structural evidence, from rapid, live-cell, three-dimensional imaging with confirmation from high-resolution electron microscopy tomography of samples preserved by high-pressure freezing and freeze substitution, unambiguously showing that from prometaphase to telophase of mammalian cells, most of the ER is organized as extended cisternae, with a very small fraction remaining organized as tubules. In contrast, during interphase, the ER displays the familiar reticular network of convolved cisternae linked to tubules.

The Small G-protein Arf6GTP Recruits the AP-2 Adaptor Complex to Membranes

The small GTP-binding protein ADP-ribosylation factor 6 (Arf6) is involved in plasma membrane/endosomes trafficking. However, precisely how the activation of Arf6 regulates vesicular transport is still unclear. Here, we show that, in vitro, recombinant Arf6GTP recruits purified clathrin-adaptor complex AP-2 (but not AP-1) onto phospholipid liposomes in the absence of phosphoinositides. We also show that phosphoinositides and Arf6 tightly cooperate to translocate AP-2 to the membrane. In vivo, Arf6GTP (but not Arf6GDP) was found associated to AP-2. The expression of the GTP-locked mutant of Arf6 leads to the plasma membrane redistribution of AP-2 in Arf6GTP-enriched areas. Finally, we demonstrated that the expression of the GTP-locked mutant of Arf6 inhibits transferrin receptor internalization without affecting its recycling. Altogether, our results demonstrated that Arf6GTP interacts specifically with AP-2 and promotes its membrane recruitment. These findings strongly suggest that Arf6 plays a major role in clathrin-mediated endocytosis by directly controlling the assembly of the AP-2/clathrin coat.

Clathrin Adaptors Really Adapt

The clathrin pathway is the principal route for receptor-mediated endocytosis and growth factor downregulation. Heterotetrameric clathrin adaptors directly link the clathrin coat with cargo transmembrane proteins that are sorted into coated pits and vesicles. A paper in this issue of Cell describes the atomic structure of the adaptor-protein 2 (AP-2) core, the portion that makes contacts with the membrane and cytosolic tails of cargo proteins.

Membrane fission by dynamin: what we know and what we need to know.

The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.