Abstract
Lysosomes are essential organelles required for breakdown of endocytic and biosynthetic cargo, pathogen killing and autophagy. In most cells, lysosomes are typically small punctate structures. By contrast, innate immune cells like macrophages and dendeitic cells that have been exposed to bacterial lipopolysaccharids (LPS) exhibit strikingly tubular lysosomes (TLs) and lysosome-related major histocompatility class II (MHCII) compartments (MIIC), respectively. TLs are suggested to play a role in phagosome maturation and retention of fluid-phase endocytic uptake in activated macrophages. In addition, the dendritic cell tubular MIIC (tMIIC) may be involved in antigen presentation. Since remarkably little was known about how tubular lysosomes form, I took to investigate the molecular requirements for this process in macrophages and dendritic cells and present my findings in this thesis. Here I confirm that microtubules are necessary as a template for lysosome tabulation, along with dynein and kinesin microtubule-dependendent motors. We were first to identify molecular components necessary for lysosome tabulation; TL biogenesis required the concerted action of the Ar18b GTPasem along with its effector SKIP, a kinesin adaptor proteins for dynein and kinesin, respectively. Importantly, we observed that TLs are highly dynamic structures whereas punctate lysosomes are conspicuously more static. I also present evidence that mTOR, a lysosomal protein kinase, is required for LPS-induced TL biogenesis and cell surface delivery of MHCII in macrophages and dendritic cells. First, I show that the MyD88-P13K-Akt-mTOR signaling pathway regulates LPS-induced lysosome tabulation. Second, I demonstrate that mTOR is required for anterograde lysosomal transport suggesting that this kinase may regulate tabulation and antigen presentation by modulating the microtubule-based motor activity of lysosomes. Finally I present preliminary data on the properties of tubular lysosomes compare to punctate lysosomes in an effort to characterize these organelles. Among the data presented is evidence that total lysosomal volume increases significantly upon tabulation, which may have important underlying implications in antigen sampling and processing. Overall, my work has expanded on our knowledge on our knowledge of how morphology and trafficking of lysosomes is modulated in immune cells, which may alter cell function.
Highlights
Lysosomes are essential organelles required for breakdown of endocytic and biosynthetic cargo, pathogen killing and autophagy
Cells respond to nutrient status and integrate signaling for growth through the protein kinase mTOR, which, not surprisingly, is deregulated in cancer and other diseases (Dazert & Hall, 2011; Zoncu, Sabatini, & Efeyan, 2011). mTOR itself is the catalytic subunit of at least two known complexes, mTOR complex 1 and mTORC2. mTOR is a ~280 kDa protein that bears a C-‐terminal kinase domain that highly resembles the catalytic domain of phosphatidylinositol 3-‐ kinase (PI3K)
Is mTOR localized to lysosomes as part of mTOR complex 1 (mTORC1), it is required for autophagic lysosome reformation, a process in which lysosomes are retrieved through tubular intermediates from autophagolysosomes at the end of autophagy
Summary
Table A-‐1: Absolute values of lysosome tubulation number for section 3.1. Cells (APCs). Abt1 – activator of basal transcription 1 Ag – antigen ALR – autophagic lysosome reformation APC – antigen presenting cell Arl – Arf-‐like Atg13 – autophagy-‐related protein 13 ATPase – adenosine triphosphatase BMDC – bone marrow-‐derived dendritic cell cDNA – complementary DNA CLIP-‐170 -‐ cytoplasmic inker protein of 170 kDa DC – dendritic cell deptor – DEP domain-‐containing mTOR-‐interacting protein EE – early endosome EEA1 – early endosomal antigen 1 FcγR – Fc gamma receptor FYCO1 – FYVE and coiled-‐coil domain-‐containing protein 1 FYVE -‐ Fab, YOTB, Vac, EEA1 GAP – GTPase activating protein GDI – GDP dissociation inhibitor GEF – guanine nucleotide exchange factor GTPase – guanosine triphosphatase IgG – immunoglobulin G IkB – inhibitor of kappa B IKK – IkB kinase IL-‐6 – interleukin-‐6 IRAK1/4 – interleukin-‐1 receptor-‐associated kinase 1/4 IRF3 – interferon regulatory factor 3 KIF – kinesin family member KIF5B – kinesin family member 5B / conventional kinesin / kinesn I xii. LAMP1 – lysosome-‐associated membrane protein 1 LC3 – light Chain 3 LE – late endosome LPS – lipopolysaccharide MAL – MyD88 adaptor-‐like MAMP – microbe-‐associated molecular pattern MHCII – major histocompatibility complex class II MIIC – MHCII compartment mLST8 – mammalian lethal with SEC13 8 mSin1 – stress-‐activated MAP-‐kinase interacting protein 1 MT -‐ microtubule MTOC – microtubule organizing center mTOR – mechanistic target of rapamycin mTORC1/mTORC2 – mTOR complex 1/2 MVB – multivesicular body MyD88 – myeloid differentiation primary response protein 88 NFkB – nuclear factor kappa B NPC1 -‐ Niemann-‐Pick C1 ORP1L -‐ oxysterol-‐binding protein-‐related protein 1L PepinhCtrl – control peptide PepinhMYD – peptide inhibitor of MyD88 PH – pleckstrin homology PI – phosphatidylinositol PI3K – phosphatidylinositol 3-‐kinase PIP – phosphoinositide PRAS40 – proline-‐rich Akt substrate protor1/2 – protein observed with rictor PRR – pattern recognition receptor qRT-‐PCR/qPCR – quantitative real-‐time polymerase chain reaction raptor – regulatory associated protein of mTOR Rheb – Ras homolog enriched in the brain xiii rictor – rapamycin insensitive component of TOR RILP – Rab7-‐interacting lysosomal protein SDS-‐PAGE – sodium dodecyl sulfate-‐polyacrylamide gel electrophoresis Ser/Thr – serine/threonine siRNA – small interfering RNA SKIP – SifA and kinesin-‐interacting protein TBK1 – TANK-‐binding kinase 1 tel2 – telomere length regulation protein 2 TL – tubular lysosome TLR – toll-‐like receptor tMIIC – tubular MHCII compartment TRAF6 – TNF receptor-‐associated factor 6 TRAM – TRIF-‐related adaptor molecule TRIF – TIR-‐domain containing adaptor inducing interferon-‐β tti1 – tel2-‐interacting protein 1 Ulk1 – Unc51-‐like autophagy activating kinase 1 V-‐ATPase – vacuolar H+-‐ATPase xiv
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