Lysosomes and the plasma membrane: trypanosomes reveal a secret relationship

Studies of the cell invasion mechanism of the parasite Trypanosoma cruzi led to a series of novel findings, which revealed a previously unsuspected ability of conventional lysosomes to fuse with the plasma membrane. This regulated exocytic process, previously regarded mostly as a specialization of certain cell types, was recently shown to play an important role in the mechanism by which cells reseal their plasma membrane after injury

Plasma Membrane Repair Is Mediated by Ca2+-Regulated Exocytosis of Lysosomes

AbstractPlasma membrane wounds are repaired by a mechanism involving Ca2+-regulated exocytosis. Elevation in intracellular [Ca2+] triggers fusion of lysosomes with the plasma membrane, a process regulated by the lysosomal synaptotagmin isoform Syt VII. Here, we show that Ca2+-regulated exocytosis of lysosomes is required for the repair of plasma membrane disruptions. Lysosomal exocytosis and membrane resealing are inhibited by the recombinant Syt VII C2A domain or anti-Syt VII C2A antibodies, or by antibodies against the cytosolic domain of Lamp-1, which specifically aggregate lysosomes. We further demonstrate that lysosomal exocytosis mediates the resealing of primary skin fibroblasts wounded during the contraction of collagen matrices. These findings reveal a fundamental, novel role for lysosomes: as Ca2+-regulated exocytic compartments responsible for plasma membrane repair

Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells

Similar to its role in secretory cells, calcium triggers exocytosis in nonsecretory cells. This calcium-dependent exocytosis is essential for repair of membrane ruptures. Using total internal reflection fluorescence microscopy, we observed that many organelles implicated in this process, including ER, post-Golgi vesicles, late endosomes, early endosomes, and lysosomes, were within 100 nm of the plasma membrane (in the evanescent field). However, an increase in cytosolic calcium led to exocytosis of only the lysosomes. The lysosomes that fused were predominantly predocked at the plasma membrane, indicating that calcium is primarily responsible for fusion and not recruitment of lysosomes to the cell surface

A Leishmania amazonensis ZIP family iron transporter is essential for parasite replication within macrophage phagolysosomes

Infection of mammalian hosts with Leishmania amazonensis depends on the remarkable ability of these parasites to replicate within macrophage phagolysosomes. A critical adaptation for survival in this harsh environment is an efficient mechanism for gaining access to iron. In this study, we identify and characterize LIT1, a novel L. amazonensis membrane protein with extensive similarity to IRT1, a ZIP family ferrous iron transporter from Arabidopsis thaliana. The ability of LIT1 to promote iron transport was demonstrated after expression in yeast and in L. amazonensis LIT1-null amastigotes. Endogenous LIT1 was only detectable in amastigotes replicating intracellularly, and its intracellular expression was accelerated under conditions predicted to result in iron deprivation. Although L. amazonensis lacking LIT1 grew normally in axenic culture and had no defects differentiating into infective forms, replication within macrophages was abolished. Consistent with an essential role for LIT1 in intracellular growth as amastigotes, Δlit1 parasites were avirulent. After inoculation into highly susceptible mice, no lesions were detected, even after extensive periods of time. Despite the absence of pathology, viable Δlit1 parasites were recovered from the original sites of inoculation, indicating that L. amazonensis can persist in vivo independently of the ability to grow in macrophages. Our findings highlight the essential role played by intracellular iron acquisition in Leishmania virulence and identify this pathway as a promising target for therapeutic intervention

The exocyst is required for trypanosome invasion and the repair of mechanical plasma membrane wounds

The process of host cell invasion by Trypanosoma cruzi shares mechanistic elements with plasma membrane injury and repair. Both processes require Ca2+-triggered exocytosis of lysosomes, exocytosis of acid sphingomyelinase and formation of ceramide-enriched endocytic compartments. T. cruzi invades at peripheral sites, suggesting a need for spatial regulation of membrane traffic. Here, we show that Exo70 and Sec8 (also known as EXOC7 and EXOC4, respectively), components of the exocyst complex, accumulate in nascent T. cruzi vacuoles and at sites of mechanical wounding. Exo70 or Sec8 depletion inhibits T. cruzi invasion and Ca2+-dependent resealing of mechanical wounds, but does not affect the repair of smaller lesions caused by pore-forming toxins. Thus, T. cruzi invasion and mechanical lesion repair share a unique requirement for the exocyst, consistent with a dependence on targetedmembrane delivery.The process of host cell invasion by Trypanosoma cruzi shares mechanistic elements with plasma membrane injury and repair. Both processes require Ca2+-triggered exocytosis of lysosomes, exocytosis of acid sphingomyelinase and formation of ceramide-enriche12812732sem informaçãosem informaçãoWe thank Dr D. Toomre (Yale University) for the VSVG construct, Dr W. Guo (University of Pennsylvania) for antibodies and A. Beaven and K. Class (University of Maryland) for assistance with confocal microscopy and flow cytometry, respectivel

Repair of injured plasma membrane by rapid Ca2+-dependent endocytosis

Ca2+ influx through plasma membrane lesions triggers a rapid repair process that was previously shown to require the exocytosis of lysosomal organelles (Reddy, A., E. Caler, and N. Andrews. 2001. Cell. 106:157–169). However, how exocytosis leads to membrane resealing has remained obscure, particularly for stable lesions caused by pore-forming proteins. In this study, we show that Ca2+-dependent resealing after permeabilization with the bacterial toxin streptolysin O (SLO) requires endocytosis via a novel pathway that removes SLO-containing pores from the plasma membrane. We also find that endocytosis is similarly required to repair lesions formed in mechanically wounded cells. Inhibition of lesion endocytosis (by sterol depletion) inhibits repair, whereas enhancement of endocytosis through disruption of the actin cytoskeleton facilitates resealing. Thus, endocytosis promotes wound resealing by removing lesions from the plasma membrane. These findings provide an important new insight into how cells protect themselves not only from mechanical injury but also from microbial toxins and pore-forming proteins produced by the immune system