Roman S. Polishchuk

A Gene Network Regulating Lysosomal Biogenesis and Function

Master Controller Cellular organelles allow the localized regulation of specialized processes. Under certain conditions, such as increased growth, organelles may be required to alter their function. Coordinated regulation of the gene networks required for mitochondrial and endoplasmic reticulum function has been observed. Now, Sardiello et al. (p. 473; published online 25 June) have discovered a gene network regulating the lysosome, the major organelle involved in the degradation of internalized macromolecules. Many lysosomal genes were regulated by a single transcription factor, TFEB. TFEB itself can be activated when the lysosome malfunctions and can regulate both the abundance of lysosomes found in the cell, as well as the ability to degrade complex molecules, including a mutant protein that accumulates in patients with Huntingtons disease. These results may have implications for the treatment of human lysosomal storage disorders, which are characterized by the aberrant accumulation of macromolecules causing cellular dysfunction. Coordination of the genes that regulate lysosomal biogenesis occurs via a shared sequence motif and one transcription factor. Lysosomes are organelles central to degradation and recycling processes in animal cells. Whether lysosomal activity is coordinated to respond to cellular needs remains unclear. We found that most lysosomal genes exhibit coordinated transcriptional behavior and are regulated by the transcription factor EB (TFEB). Under aberrant lysosomal storage conditions, TFEB translocated from the cytoplasm to the nucleus, resulting in the activation of its target genes. TFEB overexpression in cultured cells induced lysosomal biogenesis and increased the degradation of complex molecules, such as glycosaminoglycans and the pathogenic protein that causes Huntington’s disease. Thus, a genetic program controls lysosomal biogenesis and function, providing a potential therapeutic target to enhance cellular clearing in lysosomal storage disorders and neurodegenerative diseases.

Maintenance of Golgi structure and function depends on the integrity of ER export

The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1–COPII and Arf1–coatomer systems.

Golgi Membranes Are Absorbed into and Reemerge from the ER during Mitosis

Quantitative imaging and photobleaching were used to measure ER/Golgi recycling of GFP-tagged Golgi proteins in interphase cells and to monitor the dissolution and reformation of the Golgi during mitosis. In interphase, recycling occurred every 1.5 hr, and blocking ER egress trapped cycling Golgi enzymes in the ER with loss of Golgi structure. In mitosis, when ER export stops, Golgi proteins redistributed into the ER as shown by quantitative imaging in vivo and immuno-EM. Comparison of the mobilities of Golgi proteins and lipids ruled out the persistence of a separate mitotic Golgi vesicle population and supported the idea that all Golgi components are absorbed into the ER. Moreover, reassembly of the Golgi complex after mitosis failed to occur when ER export was blocked. These results demonstrate that in mitosis the Golgi disperses and reforms through the intermediary of the ER, exploiting constitutive recycling pathways. They thus define a novel paradigm for Golgi genesis and inheritance.

Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide.

The molecular machinery responsible for the generation of transport carriers moving from the Golgi complex to the plasma membrane relies on a tight interplay between proteins and lipids. Among the lipid-binding proteins of this machinery, we previously identified the four-phosphate adaptor protein FAPP2, the pleckstrin homology domain of which binds phosphatidylinositol 4-phosphate and the small GTPase ARF1. FAPP2 also possesses a glycolipid-transfer-protein homology domain. Here we show that human FAPP2 is a glucosylceramide-transfer protein that has a pivotal role in the synthesis of complex glycosphingolipids, key structural and signalling components of the plasma membrane. The requirement for FAPP2 makes the whole glycosphingolipid synthetic pathway sensitive to regulation by phosphatidylinositol 4-phosphate and ARF1. Thus, by coupling the synthesis of glycosphingolipids with their export to the cell surface, FAPP2 emerges as crucial in determining the lipid identity and composition of the plasma membrane.

Transcriptional Activation of Lysosomal Exocytosis Promotes Cellular Clearance

Summary Lysosomes are cellular organelles primarily involved in degradation and recycling processes. During lysosomal exocytosis, a Ca2+-regulated process, lysosomes are docked to the cell surface and fuse with the plasma membrane (PM), emptying their content outside the cell. This process has an important role in secretion and PM repair. Here we show that the transcription factor EB (TFEB) regulates lysosomal exocytosis. TFEB increases the pool of lysosomes in the proximity of the PM and promotes their fusion with PM by raising intracellular Ca2+ levels through the activation of the lysosomal Ca2+ channel MCOLN1. Induction of lysosomal exocytosis by TFEB overexpression rescued pathologic storage and restored normal cellular morphology both in vitro and in vivo in lysosomal storage diseases (LSDs). Our data indicate that lysosomal exocytosis may directly modulate cellular clearance and suggest an alternative therapeutic strategy for disorders associated with intracellular storage.

Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments

The organization of secretory traffic remains unclear, mainly because of the complex structure and dynamics of the secretory pathway. We have thus studied a simplified system, a single synchronized traffic wave crossing an individual Golgi stack, using electron tomography. Endoplasmic-reticulum-to-Golgi carriers join the stack by fusing with cis cisternae and induce the formation of intercisternal tubules, through which they redistribute their contents throughout the stack. These tubules seem to be pervious to Golgi enzymes, whereas Golgi vesicles are depleted of both enzymes and cargo. Cargo then traverses the stack without leaving the cisternal lumen. When cargo exits the stack, intercisternal connections disappear. These findings provide a new view of secretory traffic that includes dynamic intercompartment continuities as key players.

A tubular EHD1‐containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane

The Eps15 homology (EH) domain‐containing protein, EHD1, has recently been ascribed a role in the recycling of receptors internalized by clathrin‐mediated endocytosis. A subset of plasma membrane proteins can undergo internalization by a clathrin‐independent pathway regulated by the small GTP‐binding protein ADP‐ribosylation factor 6 (Arf6). Here, we report that endogenous EHD proteins, as well as transgenic tagged EHD1, are associated with long, membrane‐bound tubules containing Arf6. EHD1 appears to induce tubule formation, which requires nucleotide cycling on Arf6 and intact microtubules. Mutations in the N‐terminal P‐loop domain or deletion of the C‐terminal EH domain of EHD1 prevent association of EHD1 with tubules or induction of tubule formation. The EHD1 tubules contain internalized major histocompatibility complex class I (MHC‐I) molecules that normally traffic through the Arf6 pathway. Recycling assays show that overexpression of EHD1 enhances MHC‐I recycling. These observations suggest an additional function of EHD1 as a tubule‐inducing factor in the Arf6 pathway for recycling of plasma membrane proteins internalized by clathrin‐independent endocytosis.

Transport through the Golgi Apparatus by Rapid Partitioning within a Two-Phase Membrane System

The prevailing view of intra-Golgi transport is cisternal progression, which has a key prediction--that newly arrived cargo exhibits a lag or transit time before exiting the Golgi. Instead, we find that cargo molecules exit at an exponential rate proportional to their total Golgi abundance with no lag. Incoming cargo molecules rapidly mix with those already in the system and exit from partitioned domains with no cargo privileged for export based on its time of entry into the system. Given these results, we constructed a new model of intra-Golgi transport that involves rapid partitioning of enzymes and transmembrane cargo between two lipid phases combined with relatively rapid exchange among cisternae. Simulation and experimental testing of this rapid partitioning model reproduced all the key characteristics of the Golgi apparatus, including polarized lipid and protein gradients, exponential cargo export kinetics, and cargo waves.

Delivery of raft-associated, GPI-anchored proteins to the apical surface of polarized MDCK cells by a transcytotic pathway

Epithelial cell polarity depends on mechanisms for targeting proteins to different plasma membrane domains. Here, we dissect the pathway for apical delivery of several raft-associated, glycosyl phosphatidylinositol (GPI)-anchored proteins in polarized MDCK cells using live-cell imaging and selective inhibition of apical or basolateral exocytosis. Rather than trafficking directly from the trans-Golgi network (TGN) to the apical plasma membrane as previously thought, the GPI-anchored proteins followed an indirect, transcytotic route. They first exited the TGN in membrane-bound carriers that also contained basolateral cargo, although the two cargoes were laterally segregated. The carriers were then targeted to and fused with a zone of lateral plasma membrane adjacent to tight junctions that is known to contain the exocyst. Thereafter, the GPI-anchored proteins, but not basolateral cargo, were rapidly internalized, together with endocytic tracer, into clathrin-free transport intermediates that transcytosed to the apical plasma membrane. Thus, apical sorting of these GPI-anchored proteins occurs at the plasma membrane, rather than at 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.