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Journal ArticleDOI

Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells.

TL;DR: It is shown that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released.
Abstract: How the pore-forming protein perforin delivers apoptosis-inducing granzymes to the cytosol of target cells is uncertain. Perforin induces a transient Ca2+ flux in the target cell, which triggers a process to repair the damaged cell membrane. As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called 'gigantosomes'. Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released. After about 15 min, gigantosomes ruptured, releasing their remaining content. Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release.

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Citations
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Journal ArticleDOI
TL;DR: The current understanding of the structural, cellular and clinical aspects of perforin and granzyme biology is discussed, beginning to define and understand a range of human diseases that are associated with a failure to deliver active per forin to target cells.
Abstract: A defining property of cytotoxic lymphocytes is their expression and regulated secretion of potent toxins, including the pore-forming protein perforin and serine protease granzymes. Until recently, mechanisms of pore formation and granzyme transfer into the target cell were poorly understood, but advances in structural and cellular biology have now begun to unravel how synergy between perforin and granzymes brings about target cell death. These and other advances are demonstrating the surprisingly broad pathophysiological roles of the perforin–granzyme pathway, and this has important implications for understanding immune homeostasis and for developing immunotherapies for cancer and other diseases. In particular, we are beginning to define and understand a range of human diseases that are associated with a failure to deliver active perforin to target cells. In this Review, we discuss the current understanding of the structural, cellular and clinical aspects of perforin and granzyme biology.

785 citations

Journal ArticleDOI
TL;DR: Techniques for membrane disruption-based intracellular delivery from 1911 until the present achieve rapid, direct, and universal delivery of almost any cargo molecule or material that can be dispersed in solution.
Abstract: Intracellular delivery is a key step in biological research and has enabled decades of biomedical discoveries. It is also becoming increasingly important in industrial and medical applications ranging from biomanufacture to cell-based therapies. Here, we review techniques for membrane disruption-based intracellular delivery from 1911 until the present. These methods achieve rapid, direct, and universal delivery of almost any cargo molecule or material that can be dispersed in solution. We start by covering the motivations for intracellular delivery and the challenges associated with the different cargo types—small molecules, proteins/peptides, nucleic acids, synthetic nanomaterials, and large cargo. The review then presents a broad comparison of delivery strategies followed by an analysis of membrane disruption mechanisms and the biology of the cell response. We cover mechanical, electrical, thermal, optical, and chemical strategies of membrane disruption with a particular emphasis on their applications a...

420 citations

Journal ArticleDOI
24 Mar 2016-Cell
TL;DR: An unappreciated physical dimension to lymphocyte function is revealed and cells use mechanical forces to control the activity of outgoing chemical signals and data indicate that CTLs coordinate perforin release and force exertion in space and time.

274 citations


Cites background from "Perforin pores in the endosomal mem..."

  • ...This induces a repair response that enables granzymes to access the target cell cytoplasm, where they induce apoptosis (Keefe et al., 2005; Thiery et al., 2011)....

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  • ...Perforin pores drive target cell killing by inducing a membrane repair response that stimulates the uptake of additional perforin and granzymes (Keefe et al., 2005; Thiery et al., 2011)....

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Journal ArticleDOI
TL;DR: Collective evidence reveals membrane repair employs primitive yet robust molecular machinery, such as vesicle fusion and contractile rings, processes evolutionarily honed for simplicity and success.
Abstract: Eukaryotic cells have been confronted throughout their evolution with potentially lethal plasma membrane injuries, including those caused by osmotic stress, by infection from bacterial toxins and parasites, and by mechanical and ischemic stress. The wounded cell can survive if a rapid repair response is mounted that restores boundary integrity. Calcium has been identified as the key trigger to activate an effective membrane repair response that utilizes exocytosis and endocytosis to repair a membrane tear, or remove a membrane pore. We here review what is known about the cellular and molecular mechanisms of membrane repair, with particular emphasis on the relevance of repair as it relates to disease pathologies. Collective evidence reveals membrane repair employs primitive yet robust molecular machinery, such as vesicle fusion and contractile rings, processes evolutionarily honed for simplicity and success. Yet to be fully understood is whether core membrane repair machinery exists in all cells, or whether evolutionary adaptation has resulted in multiple compensatory repair pathways that specialize in different tissues and cells within our body.

258 citations


Cites background from "Perforin pores in the endosomal mem..."

  • ...However, perforin (287, 288) and SLO (122, 280) have also been shown to induce endocytosis into an abnormally enlarged endosomal compartment....

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References
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Journal ArticleDOI
09 Jul 1999-Science
TL;DR: Immunological synapse formation is now shown to be an active and dynamic mechanism that allows T cells to distinguish potential antigenic ligands and was a determinative event for T cell proliferation.
Abstract: The specialized junction between a T lymphocyte and an antigen-presenting cell, the immunological synapse, consists of a central cluster of T cell receptors surrounded by a ring of adhesion molecules. Immunological synapse formation is now shown to be an active and dynamic mechanism that allows T cells to distinguish potential antigenic ligands. Initially, T cell receptor ligands were engaged in an outermost ring of the nascent synapse. Transport of these complexes into the central cluster was dependent on T cell receptor—ligand interaction kinetics. Finally, formation of a stable central cluster at the heart of the synapse was a determinative event for T cell proliferation. A critical event in the initiation of the adaptive

2,988 citations

Journal ArticleDOI
Michael Forgac1
TL;DR: The acidity of intracellular compartments and the extracellular environment is crucial to various cellular processes, including membrane trafficking, protein degradation, bone resorption and sperm maturation, and the V-ATPases represent attractive and potentially highly specific drug targets.
Abstract: The acidity of intracellular compartments and the extracellular environment is crucial to various cellular processes, including membrane trafficking, protein degradation, bone resorption and sperm maturation. At the heart of regulating acidity are the vacuolar (V-)ATPases--large, multisubunit complexes that function as ATP-driven proton pumps. Their activity is controlled by regulating the assembly of the V-ATPase complex or by the dynamic regulation of V-ATPase expression on membrane surfaces. The V-ATPases have been implicated in a number of diseases and, coupled with their complex isoform composition, represent attractive and potentially highly specific drug targets.

1,355 citations

Journal ArticleDOI
04 Sep 1992-Cell
TL;DR: It is concluded that rab5 is a rate-limiting component of the machinery regulating the kinetics of membrane traffic in the early endocytic pathway.

1,286 citations

Journal ArticleDOI
TL;DR: Results suggest that the vacuolar type H(+)-ATPase plays a pivotal role in acidification and protein degradation in the lysosomes in vivo.

1,180 citations

Journal ArticleDOI
03 Dec 1999-Science
TL;DR: In this paper, the coding regions of the perforin gene of eight unrelated 10q21-22-linked FHL patients revealed homozygous nonsense mutations in four patients and missense mutations in the other four patients.
Abstract: Familial hemophagocytic lymphohistiocytosis (FHL) is a rare, rapidly fatal, autosomal recessive immune disorder characterized by uncontrolled activation of T cells and macrophages and overproduction of inflammatory cytokines. Linkage analyses indicate that FHL is genetically heterogeneous and linked to 9q21.3-22, 10q21-22, or another as yet undefined locus. Sequencing of the coding regions of the perforin gene of eight unrelated 10q21-22–linked FHL patients revealed homozygous nonsense mutations in four patients and missense mutations in the other four patients. Cultured lymphocytes from patients had defective cytotoxic activity, and immunostaining revealed little or no perforin in the granules. Thus, defects in perforin are responsible for 10q21-22–linked FHL. Perforin-based effector systems are, therefore, involved not only in the lysis of abnormal cells but also in the down-regulation of cellular immune activation.

1,126 citations