Steve Caplan

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.

Human Vam6p promotes lysosome clustering and fusion in vivo

Regulated fusion of mammalian lysosomes is critical to their ability to acquire both internalized and biosynthetic materials. Here, we report the identification of a novel human protein, hVam6p, that promotes lysosome clustering and fusion in vivo. Although hVam6p exhibits homology to the Saccharomyces cerevisiae vacuolar protein sorting gene product Vam6p/Vps39p, the presence of a citron homology (CNH) domain at the NH2 terminus is unique to the human protein. Overexpression of hVam6p results in massive clustering and fusion of lysosomes and late endosomes into large (2–3 μm) juxtanuclear structures. This effect is reminiscent of that caused by expression of a constitutively activated Rab7. However, hVam6p exerts its effect even in the presence of a dominant-negative Rab7, suggesting that it functions either downstream of, or in parallel to, Rab7. Data from gradient fractionation, two-hybrid, and coimmunoprecipitation analyses suggest that hVam6p is a homooligomer, and that its self-assembly is mediated by a clathrin heavy chain repeat domain in the middle of the protein. Both the CNH and clathrin heavy chain repeat domains are required for induction of lysosome clustering and fusion. This study implicates hVam6p as a mammalian tethering/docking factor characterized with intrinsic ability to promote lysosome fusion in vivo.

Normal T Cells Express Two T Cell Antigen Receptor Populations, One of Which Is Linked to the Cytoskeleton via ζ Chain and Displays a Unique Activation-dependent Phosphorylation Pattern

The TCR couples antigen recognition and the transmission of activation signals. We report the expression of two TCR populations on the surface of T lymphocytes, one of which is linked to the cytoskeleton via the ζ chain. We also demonstrate that assembly of the CD3 subunits with cytoskeleton-associated ζ is necessary for their maximal localization to the cytoskeleton. The potential significance of these two receptor forms is underscored by differences observed in non-activated T cells; while detergent-soluble phosphorylated ζ appears as a 21-kDa protein, phosphorylated cytoskeleton-associated ζ appears as a 16-kDa form. This dichotomous phosphorylation pattern is rigidly maintained following activation, although each of the receptor populations undergoes different activation-dependent modifications: 1) levels of soluble phosphorylated 21-kDa ζ are enhanced, while phosphorylated 16-kDa cytoskeleton-associated ζ exhibits little change; 2) soluble non-phosphorylated 16-kDa ζ translocates to the cytoskeleton; 3) activation-dependent ubiquitinated ζ forms localize to both fractions, albeit with different kinetics. We also show that the protein tyrosine kinase Lck undergoes activation-dependent modifications and translocates to the cytoskeleton. The phosphorylation profiles of the dichotomous TCR populations in both non-activated and activated lymphocytes suggest that each population could regulate distinct cellular functions, possibly by select intermolecular associations.

Searching for significance in TCR–cytoskeleton interactions

Two T-cell receptor (TCR) populations are expressed on T cells; one is linked to the cytoskeleton via its zeta chain. These cytoskeleton-linked receptors (30-40% of the total number of TCRs) might be important in TCR-mediated signaling and/or concurrent events. Here, differences between the two populations are summarized, and new data are examined to speculate on the functional significance of cytoskeleton-linked TCRs.

Multisubunit receptors in the immune system and their association with the cytoskeleton: In search of functional significance

Various multisubunit receptors of the immune system share similarities in structure and induce closely related signal transduction pathways upon ligand binding. Examples include the T cell antigen receptor (TCR), the B cell antigen receptor (BCR), and the high-affinity receptor for immunoglobulin E (FcεRI). Although these receptors are devoid of intrinsic kinase activity, they can associate with a similar array of intracellular kinases, phosphatases and other signaling molecules. Furthermore, these receptor complexes all form an association with the cytoskeletal matrix. In this review, we compare the structural and functional characteristics of the TCR, BCR and FcεRI. We examine the role of the cytoskeleton in regulating receptor-mediated signal transduction, as analyzed in other well-characterized receptors, including the epidermal growth factor receptor and integrin receptors. On the basis of this evidence, we review the current data depicting a cytoskeletal association for multisubunit immune system receptors and explore the potential bearing of this interaction on signaling function.

Radioiodination of Cellular Proteins

Despite the advances of non‐radioactive methods of protein labeling, radioiodination remains an important tool for studying proteins. This unit discusses several commonly used methods for radioiodination of proteins for various purposes. Included are several simple, reliable protocols for radioiodination of cell surface proteins on live cells, radioiodination of purified, solubilized membrane proteins, and radioiodination of purified soluble proteins.

Characterization and purification of a mycoplasma membrane-derived macrophage-activating factor

A highly hydrophobic component derived from the membrane ofMycoplasma capricolum has been characterized, purified and assessed for its ability to activate macrophages to tumor cytotoxicity. Initially, crude membranes were evaluated for their solubility in a wide range of solvents. Despite differential solubility in the various solvents, the mycoplasma membranes retained their ability to potentiate macrophage tumor cytotoxicity. Mycoplasma membranes were further characterized by appraising their macrophage-activating ability subsequent to various chemical treatments: cleavage of ester and thioester bonds, oxidation of vicinal hydroxyl groups, and exposure to a broad range of pH. Only strong alkaline treatment (pH>12) caused a reduction in mycoplasma membrane activity: all other chemical treatments were inconsequential. With potential therapeutic applications in mind, mycoplasma membranes were subjected to various physical treatments including heating, freezing/thawing, sonication, lyophilization and storage. The ability of the membranes to induce macrophage activation was stably maintained following all these treatments. Purification of membranes was initiated by a chloroform/methanol lipid extraction. Macrophage-activating ability was found predominantly in the interphase. Proteolytic cleavage with trypsin increased specific activity at least sixfold. Trypsinized fractions were solubilized in 2-chloroethanol and gel filtration was performed on a hydroxylated Sephadex LH-60 column. The active fraction from this column had a further tenfold increase in specific activity. Subsequent rounds of reverse-phase HPLC on this fraction yielded three to four peaks absorbing at 280 nm, of which only one had macrophage-activating ability.

The cytoskeleton-associated TCR ζ chain is constitutively phosphorylated in the absence of an active p56lck form

The TCR recognizes peptide‐MHC complexes and transmits activation signals leading to cellular responses. We have previously characterized two TCR populations expressed on the T cell surface; one is linked to the cytoskeleton via a detergent‐insoluble cytoskeleton‐associated ζ (cska‐ζ) chain, while the other is detergent soluble and not linked to the cytoskeleton. The cska‐ζ form displays unique properties: it is constitutively phosphorylated, does not undergo hyperphosphorylation upon TCR stimulation as opposed to its non‐cytoskeleton‐associated counterpart (non‐cska‐ζ) and it maintains a molecular mass of 16 kDa. It is well established that p56lck and possibly p59fyn are responsible for the generation of the 21 / 23‐kDa phosphorylated detergent‐soluble ζ form. We now demonstrate that the posphorylation of cska‐ζ does not require the activity of p56lck. We also show that although Lck does not phosphorylate cska‐ζ in vivo, it retains the capacity to phosphorylate cska‐ζ in vitro. Moreover, differences in ζ‐associated kinase activity were detected for non‐cska‐ζ and cska‐ζ. Our results indicating that different kinases phosphorylate the two ζ forms are consistent with a growing consensus that each TCR form may regulate distinct cellular functions.