Programa de Doctorado en Biociencias Moleculares
ROLE OF VMP1 IN MEMBRANE TRAFFICKING
Tesis Doctoral
Luis Carlos Tábara Rodríguez
Madrid 2018
FACULTAD DE MEDICINA
UNIVERSIDAD AUTÓNOMA DE MADRID
ROLE OF VMP1 IN MEMBRANE TRAFFICKING
Memoria presentada por el Licenciado en Biología Luis Carlos Tábara Rodríguez
para optar al grado de Doctor por la Universidad Autónoma de Madrid
Director de Tesis:
Dr. Ricardo Escalante Hernández
Instituto de Investigaciones Biomédicas “Alberto Sols” (CSIC/UAM)
FACULTAD DE MEDICINA
UNIVERSIDAD AUTÓNOMA DE MADRID
Don Ricardo Escalante Hernández, Científico Titular del Consejo Superior de Investigaciones Científicas en el Instituto de Investigaciones Biomédicas “Alberto Sols”
(CSIC/UAM)
CERTIFICA:
Que Don Luis Carlos Tábara Rodríguez, con DNI 71446529-L, licenciado en Biología, ha realizado bajo mi dirección en el Instituto de Investigaciones Biomédicas “Alberto Sols”
(CSIC/UAM), el trabajo titulado:
ROLE OF VMP1 IN MEMBRANE TRAFFICKING
Dicho trabajo reúne las condiciones requeridas por la legislación vigente, así como la originalidad y calidad científica necesarias para poder ser presentado y defendido con el fin de optar al grado de Doctor por la Universidad Autónoma de Madrid.
Madrid, a 25 de Mayo de 2018
Fdo: Ricardo Escalante Hernández Director de Tesis
Departamento de Modelos Experimentales de Enfermedades Humanas Instituto de Investigaciones Biomédicas “Alberto Sols” (CSIC/UAM) Calle Arturo Duperier 4
28029 Madrid
Esta tesis doctoral ha sido realizada gracias a una beca FPU (Formación de Profesorado Universitario) concedida en Septiembre de 2014 por el Ministerio de Educación, Cultura y Deporte a Luis Carlos Tábara Rodríguez.
“La cosa más hermosa que podemos experimentar es el misterio. Es la fuente de toda arte y toda ciencia”
Albert Einstein
Este trabajo no hubiera sido posible sin un buen número de personas. Me gustaría agradecer a todos ellos la paciencia que han tenido conmigo.
Gracias a mi director de tesis Ricardo, por haberme dado la oportunidad de hacer la tesis en su laboratorio y haberme guiado tan bien durante los primeros pasos de mi carrera científica. Gracias especialmente por mostrarse siempre dispuesto a hablar, a discutir y a “dejarme” probar ideas locas. Sinceramente, creo que hay pocos lugares en el mundo mejores para hacer una tesis.
Como siempre apuro hasta el último momento para hacer estas cosas (deposito la tesis en 2 horas, verídico), voy a dar las gracias en general a todo el mundo para no olvidarme de nadie. Gracias a mis compañeros de laboratorio estos años por las comidas, simpatía, discusiones y celebraciones. Por mostrarse siempre dispuestos a echar una mano y hablar de cualquier tema. A todos, muchas gracias de verdad.
Gracias a la gente del IIB por ser tan simpática y llevadera. Sería imposible citar a todos. Han sido unos años fantásticos tanto en lo científico como en lo personal.
Gracias a mis padres y a mi hermana por apoyarme siempre en los momentos donde la duda sobre el futuro y el pesimismo se ciernen sobre mí. Gracias por demostrarme que hay que vivir el día a día. Carpe diem!
SUMMARY / RESUMEN
SUMMARY
VMP1 is an Endoplasmic Reticulum (ER) multispanning transmembrane protein of unknown function that is highly conserved among metazoan. To shed light on the molecular function of VMP1, we have used mammalian cell culture as cellular model and state of art confocal microscopy techniques as the main experimental approach.
In this work, we show that VMP1 accumulates at dynamic ER-subdomains enriched in phospholipid synthesizing enzymes. Interestingly, VMP1 regulates the distribution and dynamics of these enzymes along the ER tubules. Moreover, these subdomains are intimately associated with other organelles such as mitochondria, endosomes, lipid droplets, peroxisomes and autophagosomes, indicating that VMP1 is a new molecular component of multiple ER-Membrane Contact Sites (MCS). Strikingly, MCS between ER and other organelles are enlarged in the absence of VMP1, suggesting that VMP1 regulates the length and function of ER-contact sites rather than acting as a tether. Furthermore, lipid membrane homeostasis of other organelles is compromised after VMP1 depletion, which leads to pleiotropic defects in organelle structure and function. These data strongly suggest that VMP1 function may be essential in maintaining a correct lipid trafficking between phospholipid enriched-ER subdomains and other organelles at MCS by regulating the distribution of the phospholipid synthesizing enzymes along the ER network.
RESUMEN
VMP1 es una proteína transmembrana de Retículo Endoplásmico (RE) de función desconocida que se encuentra altamente conservada entre los metazoos. Con el fin de arrojar luz sobre la función molecular de VMP1, hemos usado células de mamífero como modelo celular y diversas técnicas basadas en microscopía confocal como principal abordaje.
Nuestros resultados demuestran que VMP1 se acumula en ciertos subdominios del RE dónde también lo hacen distintas enzimas implicadas en la síntesis de fosfolípidos. De manera interesante, VMP1 controla la correcta distribución de estas enzimas a lo largo del entramado reticular. También hemos descubierto que los subdominios de RE enriquecidos en VMP1 se encuentran estrechamente asociados con otros orgánulos como la mitocondria, endosomas, gotas lipídicas, peroxisomas y autofagosomas, lo que indica que VMP1 es un componente nuevo y común en diversos sitios de contacto ER-orgánulo ("membrane contact sites", MCS en sus siglas en inglés). En este sentido, cabe señalar que la ausencia de VMP1 incrementa la longitud de los MCS, lo que sugiere que la proteína VMP1 no es la responsable directa del contacto sino que actúa como un regulador del tamaño y de la función del mismo. De modo muy interesante, el silenciamiento genético de VMP1 ocasiona defectos en la homeostasis lipídica de otras membranas celulares lo que lleva a defectos generalizados en la estructura y función de diversos orgánulos. Estos datos sugieren que la función de VMP1 es necesaria para mantener el tráfico lipídico entre el RE y otros orgánulos a través de regular la correcta distribución de las enzimas de síntesis de fosfolípidos en el entramado reticular.
INDEX
INDEX
ABBREVIATIONS ... 3
1. INTRODUCTION ... 7
1.1 VMP1 structure ... 7
1.2 VMP1 in mammalian cells ... 8
1.2.1 Autophagosome biogenesis in mammalian cells ... 8
1.2.2 VMP1 is required for autophagosome biogenesis ... 8
1.2.3 VMP1 in human diseases ... 9
1.3 VMP1 in simple models: clues from model organisms ... 10
1.3.1 VMP1 in Dictyostelium discoideum ... 10
1.3.2 VMP1 in Chlamydomonas reinhardtii ... 11
1.3.4 VMP1 in Caenorhabditis elegans ... 12
1.3.5 VMP1 in Drosophila melanogaster ... 13
1.4 Current knowledge about VMP1. Concluding remarks ... 13
1.5 The Endoplasmic Reticulum in mammalian cells... 13
1.5.1 Endoplasmic Reticulum structure ... 13
1.5.2 Proteins implicated in ER structure ... 14
1.6 Membrane Contact Sites between the ER and other organelles ... 16
1.6.1 General characteristics of ER-contact sites ... 16
1.6.2 Lipid transport at Membrane Contact Sites ... 17
1.6.2.1 Lipid trafficking at ER-isolation membrane contact sites ... 20
1.6.2.2 Lipid trafficking at ER-mitochondria contact sites ... 21
1.6.2.3 Lipid trafficking at ER-endosome contact sites ... 23
1.6.2.4 Lipid trafficking at ER-Golgi contact sites ... 24
1.6.2.5 Lipid trafficking at ER-lipid droplet contact sites ... 25
2. OBJECTIVES... 29
3. MATERIALS AND METHODS ... 33
3.1 Plasmids and DNA constructs ... 33
3.2 Culture of mammalian cells ... 35
3.3 DNA transfection ... 35
3.3.1 Transfection of DNA plasmids ... 35
3.3.2 Knockdown by RNAi ... 36
3.4 Autophagic and endocytic assays ... 36
3.4.1 Autophagic assays: ... 36
3.4.2 Endocytic assays: ... 37
3.5 Proteomic techniques ... 37
3.5.1 Cell lysis and Western Blot ... 37
3.5.2 Cell fractionationation ... 38
3.5.3 Immunoprecipitation assays ... 39
3.6 Immunocytochemistry and confocal microscopy ... 39
3.6.1 Molecular probes and treatments used for confocal microscopy experiments ... 40
3.6.2 Super-resolution microscopy ... 41
3.7 Electron microscopy ... 42
3.8 Image analysis ... 43
3.8.1 Confocal analysis ... 43
3.8.2 Electron microscopy analysis ... 44
3.9 Statical analysis ... 44
4. RESULTS ... 47
4.1 VMP1 is an Endoplasmic Reticulum (ER) resident protein ... 47
4.2 VMP1 dynamics along the ER network ... 49
4.3 VMP1 accumulates at specific ER subdomains enriched in phospholipid synthesizing enzymes ... 50
4.4 VMP1 and Membrane Contact Sites (MCS) ... 54
4.5 Dynamics of VMP1 subdomains at Membrane Contact Sites ... 56
4.6 Depletion of VMP1 affects the homeostasis and function of multiple organelles... 60
4.6.1 Autophagosome formation is impaired in the absence of VMP1 ... 64
4.6.2 Lipid droplet and ER homeostasis is compromised in VMP1-depleted cells ... 66
4.6.3 Mitochondrial architecture is severely affected in the absence of VMP1 ... 68
4.6.4 Endosomal trafficking is delayed in VMP1 depleted cells ... 70
4.7 Lipid trafficking at ER-endosome contact sites is altered in the absence of VMP1 ... 74
5. DISCUSSION ... 83
5.1 VMP1 localizes at ER subdomains enriched in phospholipid synthesizing enzymes ... 83
5.2 VMP1 regulates the dynamics of phospholipid synthesizing enzymes along the ER ... 85
5.3 VMP1, a new player at ER- Membrane Contact Sites ... 86
5.3.1 Characteristics of Membrane Contact Sites I ... 86
5.3.2 Characteristics of Membrane Contact Sites II ... 87
5.3.3 Characteristics of Membrane Contact Sites III ... 87
5.3.3.1 Endoplasmic reticulum defects... 88
5.3.3.2 Mitochondrial defects ... 88
5.3.3.4 Lipid droplet defects ... 89
5.3.3.5 Endosomal defects ... 90
5.3.3.6 Golgi defects ... 92
5.3.4 Commentary about VMP1 function in MCS ... 92
5.4 VMP1 and autophagy, a long story finally clarified ... 93
5.5 Future directions ... 95
5.5.1 VMP1 interactors ... 95
5.5.2 VMP1 topology ... 95
5.5.3 Conserved motifs In VMP1 sequence ... 96
6. CONCLUSIONS ... 101
7. REFERENCES ... 107
8. ANNEXES ... 129
8.1 SUPPLEMENTARY INFORMATION ... 129
8.2 PUBLICATIONS RELATED WITH THIS WORK ... 130
8.3 OTHER PUBLICATIONS ... 130
ABBREVIATIONS
3 ABBREVIATIONS
ATG autophagy related BFP blue fluorescent proteins BSA bovine serum albumine
CDP DAG cytidine diphosphate diacylglycerol
CEPT1
choline/ethanolaminephosphotransferase 1 CERT ceramide transfer protein
CI-M6PR cation-independent-manosse 6 phosphate receptor
CL cardiolipin CNX calnexin CQ chloroquin DAG diacylglycerol
DFCP1 double fyve containing protein 1 DGDG digalactosyldiacylglycerols
DMEM dulbecco's modified eagle medium DMSO dimethyl sulfoxide
DTT dithiothreitol
EBSS earle's balanced salt solution EE early endosome
EEA1 early endosome antigen EGF epidermal growth factor
EGFR epidermal growth factor receptor
EMC endoplasmic reticulum membrane protein complex
EPG-3 ectopic P granules protein 3 ER endoplasmic reticulum
ERMES endoplasmic reticulum-mitochondria encounter structure
FFAT two phenylalanines (FF) in an acidic tract
FIP200 FAK family kinase-interacting protein of 200 kDa
FIS1 mitochondrial fission 1 protein GFP green fluorescent protein
HRS hepatocyte growth factor-regulated tyrosine kinase substrate
IM isolation membrane KMS kill me softly LC3 light chain 3 LD lipid droplet
LDL low density lipoprotein LE late endosome
LPP2 lipid phosphate phosphatase 2 LTPs lipid transfer protein
MAMs mitochondria associated membranes MCS membrane contact sites
MFN2 mitofusin 2 MT-Red mitotracker-red NC nocodazole
NE nuclear envelope
NIR2 Pyk2 N-terminal domain-interacting receptor 2
NPC niemann pick type C OA oleic acid
ORP oxysterol-related protein OSBP oxysterol-binding protein PA phosphatidic acid
4 PBS phosphate-buffered saline
PC phosphatidylcholine PCR polymerase chain reaction PE phosphatidylethanolamine PFA paraformaldheide PG phosphatidylglycerol PH pleckstrin-Homology
PIS1 phosphatidylinositol synthase 1 PITP phosphatidylinositol transfer protein PL phospholipid
PM plasma membrane PS phosphatidyilserine PtdIns phosphatidylinositol
PtdIns3K phosphatidylinositol 3 kinase PtdIns3P phosphatidylinositol 3 phosphate PtdIns4K phosphatidylinositol 4 kinase PtdIns4P phosphatidylinositol 4 phosphate RAB ras-associated Binding
RFP red fluorescent protein RNAi RNA interferance RT room temperature RTN reticulon
SAC1 suppressor of actin mutations 1-like protein
SAR1 secretion-associated RAS-related protein 1
SD standard desviation SDS sodium dodecyl sulfate
SMP synaptotagmin-like mitochondrial-lipid- binding domain
SNX2 syntaxin2
SR-SIM super-resolution structured illumination microscopy
STARD StAR-related lipid-transfer TAG triacylglycerol
TANGO5 transport and golgi organization 5 TBS-T tris buffered saline with tween TEM transmission electron microscopy TGN transgolgi
TIM translocase of the inner membrane TOM translocase of the outer membrane TVP38 TLG2-vesicle protein of 38 kDa ULK1 unc-51-like kinase 1
VAP vamp associated protein
vCLAMP vacuole and mitochondria patch
VDAC voltage-dependent anion-selective channel protein
VMP1 vacuole membrane protein 1 WB western blot
WIPI2 WD repeat domain phosphoinositide- interacting protein 2
WT wild Type
INTRODUCTION
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1. INTRODUCTION
1.1 VMP1 structure
VMP1 (Vacuole Membrane Protein 1) is an Endoplasmic Reticulum (ER) resident protein of 406 aminoacids that is largely conserved among metazoans but it is absent in yeast 1 2 3 4 5. Hydrophobic analysis of the protein predicts that VMP1 is a multispanning membrane protein of six transmembrane helices. Topological studies in plants 1 have revealed relevant structural aspects of the protein such as the presence of a cytoplasmic loop between the second and third transmembrane helix and the cytoplasmic localization of both N- and C- terminus regions (Figure 1). Importantly, VMP1 harbors a C-terminal lysine motif which is implicated in the retrieval of the protein from the Golgi 6. Moreover, a snare-like domain 7 of unknown function is present in the third transmembrane helix. The high conservation of the amino-acid sequence suggests that human VMP1 likely shares the same topology with six transmembrane regions.
The final region of the loop is specially conserved in all metazoans suggesting that it may be important for VMP1 function.
Despite it was described for the first time in 2002 5, literature about VMP1 is relatively scarce.
During the last fifteen years, VMP1 has been studied in several model organisms but its molecular function remains unknown. In the first part of this introduction, I present the current knowledge about VMP1 function in different cellular models with special emphasis in mammalian cells (which is the model system used in this thesis).
Figure 1 l VMP1 topology. Proposed topology for VMP1 in plants. Similar topology may exist in other organisms because of the high conservation of the protein sequence. No functional domains are found in the protein but the cytoplasmic loop is very conserved suggesting that it may be important for VMP1 function.
8 1.2 VMP1 in mammalian cells
Autophagy is an intracellular degradation mechanism that delivers cytoplasmic proteins and organelles to the lysosome 8 9. Remarkably, until our first publication presented in this thesis 10, autophagy was the only function described for VMP1 in mammalian cells 4 11 12.
1.2.1 Autophagosome biogenesis in mammalian cells
Autophagy is mediated by a unique organelle called autophagosome, which is assembled at the endoplasmic reticulum (ER) from a precursor structure named isolation membrane or IM 13
14. Nucleation of the IM requires the ULK1-FIP200 complex and the transmembrane protein ATG9 15 16 17. When autophagy is activated by starvation, ULK1 is recruited to specific ER subdomains enriched in PIS1 (phosphatidylinositol synthase) 16 18. Once there, ULK1 recruits the PtdIns3K complex through its specific autophagic partner ATG14 to generate the PtdIns3P (phosphatidylinositol 3-phoshate) enriched omegasome, an ER-derived structure that serves as a cradle for IM elongation 19 20 21 22. In this connection, it has been recently proposed that ATG9-containing vesicles nucleate the initial IM 15. The relationship between PIS1 subdomains and the omegasome is intriguing since it is possible that PtdIns (phosphatidylinositol) synthesized in these subdomains feed PtdIns3K enzyme to generate PtdIns3P at the omegasome formation site 18. Once the omegasome is formed, ULK1 initiation complex is translocated from the ER to IMs in a PtdIns/PtdIns3P dependent manner. In this way, it has been proposed that transfer of PtdIns/PtdIns3P from PIS1-enriched ER subdomains to the IM (probably by lipid transfer proteins) is necessary to stabilize the ULK1 complex at the IM 16. Later, PtdIns3P produced by PtdIns3K recruits WIPI2 which, in turn, recruits the ATG12–ATG5–
ATG16L1 complex that facilitates the transfer of LC3 from ATG3 to phosphatidylethanolamine (PE) at the edge of the IM for continue with the elongation process 23. During the elongation process, the IM will receive membranes from different sources to form the autophagosome 14
24 2526 27. Once the autophagosome is completely formed, it will detach from the ER and will be transported toward the lysosome to complete the degradative process 28.
1.2.2 VMP1 is required for autophagosome biogenesis
Confocal studies have shown that mammalian VMP1 accumulates at specific puncta along the ER. Interestingly, these puncta transiently associate with early autophagic markers during autophagosome formation 15 29 30. In this way, Itakura et al. 30 found that VMP1 puncta are also generated in FIP200 knock-out cells suggesting that VMP1 could be an upstream factor of the ULK1 complex. However, after VMP1 depletion not only ULK1 but also WIPI2, ATG16 and LC3 puncta are still formed indicating that VMP1 is not necessary for autophagy induction and for
9
the recruitment of autophagic proteins 4 30. Intriguingly, autophagic structures accumulate and become abnormally large in VMP1 silenced cells suggesting that VMP1 function is essential for the proper turnover of autophagic proteins during autophagosome biogenesis. As expected, LC3 turnover and autophagic flux is blocked demonstrating that autophagic degradation is impaired in the absence of VMP1 30. TEM (Transmission Electron Microscopy) analysis confirmed the accumulation of IMs in VMP1 depleted cells 31. Furthermore, these IMs are starvation-dependent and positive for LC3 indicating that they are immature autophagic structures that are not able to elongate into complete autophagosomes 31.
As mentioned, the omegasome is an ER-derived structure enriched in PtdIns3P that serves as a guiding platform for the nascent IM 21. Remarkably, the omegasome marker DFCP1 19 also accumulates and becomes aberrant in VMP1 silenced cells suggesting that defective autophagy may arise from defects in ER structure. Moreover, these abnormal DFCP1 structures colocalize with the autophagosomal marker LC3 indicating that the accumulation of LC3 is primarily caused by an increased number of omegasomes that presumably cannot stabilize properly the IMs during the elongation process.
How VMP1 regulates the turnover of autophagic proteins from the ER is still a mystery. The aberrant morphology of the omegasome in VMP1-depleted cells suggests that VMP1 may have a function in the remodeling of ER membranes during omegasome formation. Thus, functional relationship between VMP1 and other ER domains involved in omegasome biogenesis (such as PIS1 subdomains) will be addressed in this thesis.
1.2.3 VMP1 in human diseases
To date, mutations in VMP1 gene have not been associated with any disease. However, VMP1 levels are altered in some pathological conditions. For example, VMP1 expression is upregulated during both acute and chronic pancreatitis 32 33 34 35 36. Similarly, VMP1 upregulation has been found in ovarian 37 and pancreatic tumors 38. Nonetheless, VMP1 function in cancer seems to be cell type dependent since downregulation of VMP1 correlates with aggressive properties of both colorectal 39 40 and hepatocellular carcinomas 41. Moreover, genomic rearrangements involving VMP1 gene has been also associated with cancer. For example, RPS6KB1-VMP1 gene fusion is a recurrent event in both esophageal adenocarcinoma
42 and primary breast cancer 43. Interestingly, in-vitro expression of this chimera results in aberrant localization of the protein and causes defective autophagy 42.
10
Finally, VMP1 was also identified in a high-throughput study as a gene whose deletion confers resistance to the lytic infection of several viruses (HIV among them) in human macrophages 44.
1.3 VMP1 in simple models: clues from model organisms
The protein sequence of VMP1 is highly conserved among metazoans (Figure 2), and thus model organisms provide a useful tool to study the molecular function of this protein.
1.3.1 VMP1 in Dictyostelium discoideum
Among all model systems, D. discoideum is the one in which VMP1 function has been studied more extensively. Similar to other organisms, VMP1 mutant cells are not able to survive under starvation conditions indicating that autophagy is impaired 12. In agreement with this, disruption of VMP1 gene leads to the formation of aberrant and persisting structures of ATG18 (WIPI2 homologous) irrespective of the growth conditions 45. The PtdIns3P marker 2xFYVE colocalizes with these abnormal ATG18 structures in VMP1 mutant cells suggesting that ATG18 accumulation may be caused by an uncontrolled flux of PtdIns3P from the ER 45. Interestingly, both ATG18 and PtdIns3P accumulations are not present in double knockout cells for ATG1 (the ULK1 homologous) and VMP1 confirming that the abnormal PtdIns3P flux emerge from the ER downstream of the ATG1 complex 45. Therefore, it has been proposed that VMP1 may regulate PtdIns3P levels at the ER during autophagosome formation.
Besides autophagy, multiple defects are observed in Dictyostelium null mutants for VMP1 2. One of the most distinguishable defects is the inability of mutant cells to cope with hyposmotic stress due to defects in contractile vacuole biogenesis. In addition, VMP1 null cells are not able to swell during hyposmotic stress and rapidly burst, suggesting that plasma membrane (PM) stability is compromised. Moreover, ER and Golgi integrity are affected since they are fragmented 2. Consistently, protein secretion (which depends on ER-Golgi communication) is also compromised in VMP1 mutant cells.
VMP1 deficient cells are not able to perform macropinocytosis and consequently are not viable for more than 2-3 days in axenic media 2 45. In this connection, appropriate levels of PtdIns3P
Figure 2 l VMP1 in model organisms. e-Value of VMP1-orthologs in different metazoan groups. The most similar proteins to the human VMP1 are listed in order, from the highest identity to the lowest.
VMP1 protein sequence is highly conserved across evolution.
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are not only important for autophagosome formation but also during endosomal trafficking of macropinocytic vacuoles, as PtdIns3P regulates the turnover of different effectors on endosomal membranes 46 47 48. During autophagy, PtdIns3P generation depends on ATG1 complex 49 50. As mentioned before, double knock-out of ATG1 and VMP1 suppress the aberrant accumulation of ATG18 and PtdIns3P at ER membranes. Surprisingly, this double knock-out also rescues the macropinocytosis defect observed in the simple VMP1 mutant strain, 45 suggesting that the abnormal and uncontrolled autophagy-related flux of PtdIns3P at the ER might compromise general PtdIns3P homeostasis in the cell and thus affecting other autophagy-independent processes.
Together, all these data demonstrate that mutant cells for VMP1 in Dictyostelium display multiple defects in membrane trafficking and organelle biogenesis. Interestingly, expression of human VMP1 rescues all defective phenotypes of the VMP1 mutant strain demonstrating that VMP1 polyvalent function is evolutionarily conserved 2.
1.3.2 VMP1 in Chlamydomonas reinhardtii
Like in Dictyostelium, knock-out cells for VMP1 in Chlamydomonas display a wide arrange of cellular defects. Defective cytokinesis is a hallmark of this mutant strain since daughter cells aberrantly attached each other when they divide. The cellular shape is also irregular compared with the oval form of WT cells, suggesting that PM homeostasis is compromised 3.
Observation of VMP1 mutant cells by electron microscopy confirms the aberrant ultrastructure of membranous
organelles 3. For example, Chlamydomonas usually have one Golgi apparatus per cell.
However, VMP1 null cells have three or four deformed Golgi apparatuses. Mitochondrial shape is also altered in the mutant strain since they are larger and have dilated cristae. Moreover, chloroplast are disorganized and mislocalized in VMP1 null cells indicating a general defect in organelle biogenesis. Interestingly, vacuoles (similar to mammalian lysosomes) were electrodense in WT cells (likely because they are plenty of membrane and cytoplasmic elements) but clear in mutant cells suggesting that autophagy may be affected like in other model organisms.
Metabolomic studies in Chlamydomonas also revealed significance differences in VMP1 mutant cells 3. For example, triacylglycerol (TAG) accumulation was the most striking alteration between WT and mutant cells and also, accordingly with the phenotypical defects, lipids involved in membrane biogenesis such as digalactosyldiacylglycerols (DGDG),
12
phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) were reduced in VMP1 null cells.
1.3.3 VMP1 in plants
Plants have two homologous of VMP1: KMS1 and KMS2. Depletion of KMS1 expression by RNAi in Arabidopsis leads to the accumulation of ER membranes in aberrant whorls and Golgi apparatus fragmentation1. Inhibition of KMS2 leads to similar phenotypes although less severe.
This could be explained by a different efficiency of the RNAi hairpins or by certain degree of overlapping functions of both proteins. Regarding plant morphology, knockdown of KMS1 causes growth defects with shorter roots than the wild type 1.
Similarly, overexpression of a truncated protein lacking the conserved cytoplasmatic loop in tobacco leaf epidermal cells caused fragmentation of the ER into swollen structures and redistribution of Golgi markers to the aberrant ER 1. As expected, protein secretion is also compromised and secretory cargos remain trapped in the distorted ER when the truncated protein is expressed 1. These results strongly suggest that KMS1/KMS2 may function in maintaining ER/ Golgi integrity and functionality in plants.
1.3.4 VMP1 in Caenorhabditis elegans
During C.elegans development, PGL granules are degraded by autophagy 51 52. EPG-3 (the homologous of VMP1 in C.elegans ) was identified in a genetic screen as a gene required for the degradation of PGL granules 4. Like other autophagic mutants, EPG-3 mutant worms display reduced survival of larvae in absence of food.
EPG-3 deficient worms accumulate PGL granules surrounded by LGG-1 (the homologous of LC3)-labeled structures. Interestingly, LGG-1 puncta are morphologically defective at all stages of the embryogenesis as they are larger and tend to form abnormal tubular structures.
Moreover, the omegasome marker DFCP1 colocalizes with these LGG-1 puncta indicating that they are early and immature autophagic structures. TEM confirmed the accumulation of IMs indicating that EPG-3/VMP1 function is necessary for the elongation and closure of autophagosomes 4. Hence, autophagic defective phenotypes found in EPG-3 mutant worms are very similar to those described for VMP1 in mammalian cells. In fact, human VMP1 rescues the aberrant accumulation of PGL granules present in EPG-3 mutants cells demonstrating that VMP1 function is conserved across metazoans 4.
13 1.3.5 VMP1 in Drosophila melanogaster
TANGO5 is the homologous protein of VMP1 in Drosophila melanogaster. There is not much information about the function of TANGO5/ VMP1 in this model organism. It has been reported that TANGO5 is required for constitutive protein secretion from the ER during the early secretory pathway since RNAi for TANGO5 results in the redistribution of the Golgi marker MannII-GFP from Golgi to ER membranes 53 54.
1.4 Current knowledge about VMP1. Concluding remarks
As described in the previous pages, VMP1 function has been implicated in a multitude of processes in different organisms. How a single protein could be involved in such a variety of processes is unknown and the molecular function of VMP1 is still a mystery.
Certainly, most of phenotypic defects found in VMP1 mutant cells are directly related with endoplasmic reticulum (ER) functionality such as the abnormal ER and Golgi structure or the impaired protein secretion from the ER 1 2 3 54. Defective autophagy 4 1230 is also likely caused by structural defects at the ER during omegasome formation 20. Additionally, it is known that the ER makes contacts with other organelles at Membrane Contact Sites (MCS). MCS are regions where the ER comes in close apposition to other endomembranes forming contact areas where lipid exchange and calcium signaling occur. In this way, dysregulation of MCS could potentially explain the wide array of defective phenotypes observed in model organisms such as the impaired macropynocitosis 2 45 (defects at ER-endosome contacts), membrane fragility 23 (defects at ER-plasma membrane contacts) or the abnormal mitochondrial ultra-structure 3 (defects at ER-mitochondria contacts).
Our hypothesis at the beginning of this work was that VMP1 could be involved in maintaining functional ER structure and dynamics in the interface between ER membranes and other organelles at MCS. Therefore, we have focused this thesis on analyzing if VMP1 could be enriched at certain ER subdomains that may regulate both ER structure and MCS functionality in mammalian cells.
1.5 The Endoplasmic Reticulum in mammalian cells 1.5.1 Endoplasmic Reticulum structure
The ER is the largest organelle in the cell and serves multiple functions including protein and lipid synthesis 55 56 57. Generally, the ER can be classified as rough ER, characterized by the presence of ribosomes, and smooth ER which is devoid of ribosomes. The presence of
14
ribosomes indicates that rough ER is functionally linked to the synthesis of proteins 58. Additionally, rough ER is also involved in protein transport to Golgi membranes via COPII vesicles 59. On the other hand, the smooth ER has been implicated in both lipid metabolism and Ca2+ homeostasis 5560.
Regarding its structure, three major ER domains can be distinguished: the nuclear envelope (NE), the juxtanuclear area (characterized by the presence of expanded ER sheets) and the peripheral zone, which is enriched in tubular-like structures (Figure 3) 566162. The NE separates the nucleus from the cytoplasm, forming a continuous with the outer nuclear membrane and the rest of the ER. The NE serves as a selective barrier to control the transport of molecules in and out of the nucleus 63 64. ER sheets, localized typically near the juxtanuclear zone, are expanded areas with little membrane curvature where intense protein translation and modification occur 61. Finally, ER tubules are highly curved membranes located predominately in the cell periphery and are involved in lipid synthesis since they contain a high concentration of lipid synthesizing enzymes 65.
1.5.2 Proteins implicated in ER structure
How the particular shape of the ER is generated and how the balance between tubules and sheets is regulated are fundamental questions in cell biology 6566. Tubule formation depends on proteins harboring reticulon homology domains (present in Reticulons/RTNs and REEPs in mammals), which consist in hydrophobic segments of around 30 aminoacids with the ability to get inserted into membranes as wedge-shaped hairpins, thus generating intense membrane curvature 67 68 69 . According to this property, the overexpression of RTNs deforms proteo- liposomes into tubules demonstrating that these proteins are sufficient to shape membranes 70
71. On the other hand, sheets show high curvature only at the edges and accordingly, RTNs are restricted to those regions. It seems that, in general, the lack of curvature is due to the
Figure 3 l Endoplasmic Reticulum (ER) structure. A COS-7 cell expressing the ER resident protein VAPA. The ER spreads into the cytosol as a complex network of sheets and tubules. ER sheets concentrate at the juxtanuclear area whereas the cell periphery is enriched in ER tubules.
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absence of reticulons, and in fact, it has been experimentally proved that ER sheets can be propagated by the depletion of RTNs and REEPs 707273.
Connecting tubules into the characteristic polygonal ER network requires membrane fusion.
Members of the Atlastin family of GTPases have been largely involved in ER fusion during the formation of tubular three-way junctions. In this way, depletion of Atlastin-3 (ATL3) or expression of dominant-negative mutants leads to the formation of long and unbranched tubules 747576. How ER junctions are maintained is unknown, but it has been suggested that they are stabilized by the protein Lunapark 777879.
Remarkably, different studies have implicated RAB proteins in the maintenance of ER structure. For example, characterization of RAB GTPases in C.elegans identified RAB5 as a protein required for efficient homotypic fusion of ER tubules 80. RAB18 and its GEF Rab3GAP have also been implicated in ER structure since the absence of both proteins alters the balance between tubules and sheets by increasing the sheet area 81. Similarly, it has been reported that RAB10 plays an active role in ER structure and tubule dynamics. Depletion of RAB10 or expression of the negative mutant form reduces ER tubule fusion efficiency and cause the spreading of the sheet zone towards cell periphery 8283. Interestingly, RAB10 colocalizes at the leading edge of growing ER tubules with the phospholipid (PL) synthesizing enzymes CEPT1 (Choline/Ethanolamine Phosphotransferase) and PIS1, which are ER resident transmembrane proteins. It is possible that PL synthesizing enzymes localize at the tubule tip to provide the necessary phospholipids for ER growth. Alternatively, the authors suggest that accumulation of PL synthesizing enzymes may facilitate the transfer of phospholipids to other organelles bounded to the tip of ER tubules 82.
The last hypothesis was initially proposed by Kim et al. 8485 who suggested that PIS1 marks an ER-derived subdomain specialized in the delivering of PtdIns to other endomembranes such as mitochondria and endosomes (Figure 4). Certainly, PtdIns-enriched ER subdomains would represent a very efficient way of deliver PtdIns to other cellular membranes at Membrane Contact Sites (MCS). Accordingly, it has been demonstrated that PtdIns synthesized at PIS1- enriched ER is transported to IMs during autophagosome biogenesis as described above 16 18. It is unknown how the delivery of PtdIns occurs from these ER subdomains but it is possible that lipid transfer proteins (LTPs) may participate in this process (Figure 4). Intriguingly, Kim et al.
demonstrated that some PIS1 puncta emerge from the ER and behave like cytoplasmic vesicles between ER tubules 84 . Eventually, these vesicles are able to return to ER tubules, likely to resupply PIS1 with its substrate CDP-DAG (CDP-diacylglycerol)84 (Figure 4). Authors suggested
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that these PIS1-enriched vesicles could also donate PtdIns to other organelles by hemifusion of apposed membranes. In this regard, the mechanisms involved in the transfer of lipids from the ER to other membranes will be discussed in detail in the last part of this introduction.
1.6 Membrane Contact Sites between the ER and other organelles 1.6.1 General characteristics of ER-contact sites
The ER forms an extensive network of Membrane contact Sites (MCS) with most intracellular organelles and the plasma membrane 868788 (Figure 5). Several studies based on tomography and electron microscopy have captured some relevant characteristics of ER-contact sites at nanometer resolution. One defining feature of ER-contact sites is that ribosomes are excluded from the ER membrane that is contact with the second organelle 89. Moreover, the ER and the partner organelle are closely apposed but don´t fuse each other (the distance varies between 10-30 nm) 909192. Importantly, MCS are stabilized by different molecular tethers that bind to both membranes 939495. In fact, organelles and the ER are so tightly tethered that the formers may drag the ER tubules as they move 87969798. For this reason, it has been proposed that ER structure and dynamics may be highly influenced by MCS 87. The length and shape of MCS are dynamic and correlate with the functional demands of the organelles, which include lipid and calcium exchange, organelle movement and fission. Particularly in this thesis we have focused on the study of lipid trafficking at ER-membrane contact sites.
Figure 4 l PIS1 define a PtdIns-enriched ER subdomain with unique characteristics. The ER resident protein PIS1 accumulates at certain regions of the ER forming punctated structures.
Intriguingly, some PIS1 puncta are able to exit the ER and behave like vesicles. It has been proposed that these structures may serve as PtdIns delivery platforms to other cellular endomembranes. CDS1/2 (CDP- Diacylglycerol Synthase) provides CDP- DAG, the substrate of PIS1. Figure adapted and modified from Kim et al. 83.
17 1.6.2 Lipid transport at Membrane Contact Sites
Membranes of eukaryotic cells are composed by a wide variety of different lipids 99100. In this way, lipid biosynthesis and transport to cellular endomembranes ensure that each organelle will have its characteristic lipid composition that supports the function of the associated proteins and signaling pathways 101.
The ER is the main site of lipid synthesis in the cell 55. Newly synthesized lipids at the ER must be delivered to different endomembranes since most organelles lack the capability to synthesize lipids de novo 102 103104. Once lipids arrive at their destined membrane, they are converted into distinct species in order to preserve organelle identity. For example, PtdIns synthesized at the ER membrane is transformed to a variety of PtdInsP in the plasma membrane by phosphorylation of the inositol ring at different positions 105 106 107. Similarly, phosphatidylserine (PS) is converted to phosphatidylethanolamine (PE) when arrives to mitochondrial membranes 108. Thus, defective lipid trafficking from the ER affects lipid homeostasis of virtually all cellular endomembranes 102.
There is compelling evidence that impaired secretory vesicular pathway does not disturb lipid trafficking between ER and other organelles 109 110 111 112 113 114 115. Therefore, non-vesicular
Figure 5 l Endoplasmic Reticulum forms Membrane Contact Sites (MCS) with other organelles. The ER expands throughout the cell into a dynamic network of sheets and tubules. Peripheral ER tubules make close contacts (<30 nm) with most cellular organelles and plasma membrane at MCS. ER- contact sites have multiple functions such as lipid trafficking, calcium exchange and organelle remodeling. Figure adapted and modified from Holthuis & Levine 121.
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mechanisms may exist to facilitate lipid trafficking between both membranes 116117118 (Figure 6). In this way, the spontaneous and passive diffusion of phospholipids from one membrane to another through the aqueous cytoplasm can occur but it is extremely slow 102 119. Lipid transport can be accounted much faster by the action of the so called Lipid Transfer Proteins (LTPs). LTPs comprise several protein families with hydrophobic domains that are able to desorbing lipid monomers from lipid bilayers and transfer them through the aqueous gap to different membranes 120121122 (Figure 6). During the last years, it has been demonstrated that LTPs may function at Membrane Contact Sites (MCS) rather than moving long distances in the cytosol 95119123. LTPs can interact with one or both membranes in different ways to perform its function through specific domains (Figure 6). In this way, several LTPs harbor a FFAT (two phenylalanine in acid tract) motif that directly interact with the ER-resident proteins VAPs (Vesicle-Associated membrane Protein (VAMP)-associated proteins) 124125. Binding of LTPs to the second membrane at MCS is usually mediated by another LTP interacting protein or by lipid binding domains such as pleckstrin homology (PH) or C2 domains, which directly bind to the second membrane 110 126 127 128 129. Hence, LTPs might be involved not only in lipid trafficking but also in the formation and stabilization of MCS.
Although lipid transport mediated by LTPs has been well established, it is clear that other non- vesicular traffic mechanisms exist at MCS. One of these alternative mechanisms involve SMP (synaptotagmin-like mitochondrial-lipid binding protein)-containing proteins, which define a
Figure 6 l Lipid trafficking mechanisms between the ER and other organelles. Lipid trafficking may occur by both vesicular (A) or non vesicular mechanisms. Spontaneous movement of lipids (B) is too slow. Lipid exchange can be facilitated by lipid transfer proteins (LTPs) (C). Importantly, LTPs preferentially act at MCS (D-F), where they rapidly move lipids by interacting with both donor and acceptor membranes. Additionally, lipid transport can be also accounted by the action of integral membrane proteins that forms hydrophobic channels between both membranes (G). Figure adapted and modified from Toulmay and Prinz 118.
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superfamily of proteins that localize at multiple MCS in both yeast and mammalian cells 130131. These proteins are able to bind lipids and form hydrophobic channels that facilitate lipid trafficking across membranes 132 133 134135 (Figure 6G). Alternatively, transient hemifusion of both membranes has also been proposed as another non-vesicular mechanism at ER-contact sites 118136137.
Remarkably, non-vesicular trafficking processes tend to take place at specific ER subdomains were lipid synthesizing enzymes concentrate 102108115138139. This localization helps to create a lipid enriched microenvironment that increases the likelihood for lipid monomers to be transferred between membranes by the driving force of lipid gradients. Moreover, specific lipid composition of the ER also determines biophysical properties of the membranes that may favor the kinetics of lipid transfer reactions at MCS 116 140. For example, higher transfer activities are associated with more fluidic and curved membranes because the interactions between the lipids and the bilayer are weaker and the detachment or absorption of the lipid to LTPs is more efficient 141142143.
Finally, it should be noted that non-vesicular trafficking at MCS tends to equilibrate the level of lipids between both membranes 102 116. Preservation of the characteristic lipid properties of each membrane is essential for maintaining active gradients and lipid exchange. Therefore, a recurrent question in the field is how the differential composition of lipids is maintained at MCS to preserve organelle identity. One proposed mechanism is known as “trapping” 144. In this way, lipids can be trapped at their destined membranes because of its electrostatic affinity for another lipids or proteins (thermodynamic trapping). Alternatively, lipids can be modified or transformed into another in the second membrane in order to maintain the unidirectional trafficking of lipids at MCS (metabolic trapping) (Figure 7).
Figure 7 l Directional lipid trafficking at MCS. Lipid synthesizing enzymes (red) concentrates at MCS to increase the likelihood of lipid transfer. Once lipid monomers arrive to their destined membrane, they can be transformed into other species (metabolic trap) or be retained by its electrostatic interaction with other proteins (thermodynamic trap). These mechanisms maintain active lipid gradients at MCS. Figure adapted and modified from Lahiri et al. 101.
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Importantly, lipid trafficking at ER-contact sites display specific characteristics depending on the organelles involved. We will describe every of them in more detail in the following pages.
1.6.2.1 Lipid trafficking at ER-isolation membranes contact sites
As described in the first part of the introduction, MCS between the ER and the IM occur at the PtdIns3P-enriched omegasome 20 21. It has been proposed that PtdIns synthesized at PIS1 subdomains may serve as substrate for the PtdIns3K complex during omegasome formation 16 (Figure 8). Then, PtdIns and PtdIns3P are transported to the IM in order to stabilize the ULK1 complex (Figure 8). Interestingly, it has been reported that other phospholipid synthesizing enzymes such as CEPT1 and PSS1 (Phosphatidylserine synthase 1) also concentrates at ER-IM contact sites 1618. Therefore, it is tempting to speculate that different phospholipids may traffic from the ER to nascent autophagosomes. Unfortunately, to date, non-vesicular lipid transport mechanisms between the ER and the IM have not been identified. Nevertheless, it has been recently published that VAPs proteins localize at ER-IM contact sites (Figure 8) 145. As commented before, VAPs proteins interact with the FFAT region of multiple LTPs at MCS 124 raising the possibility that LTPs may function in the trafficking of phospholipids between the ER and IMs. Thus, future studies will be required to decipher the identity of these LTPs.
Interestingly, it has been recently reported that the interaction of WIPI2 with the ULK1/
FIP200 complex contributes to the formation of ER-IM contact sites 146. The same authors also described that VAPs interact with FIP200, ULK1 and WIPI2 145 and that depletion of VAPs decreases the interaction between WIPI2 and ULK1/ FIP200. Therefore, these data suggest that VAPs may also have a direct role in the regulation of the autophagic machinery 145.
Figure 8 l ER-Isolation Membranes (IM) Contact Sites. PtdIns synthesized at PIS1 subdomains is transformed to PtdIns3P by the PtdIns3K complex leading to the formation of the PtdIns3P-enriched omegasome, which serve as a cradle for the nascent IM. Then, PtdIns and PtdIns3P are transported to the IM to stabilize there the ULK1 complex and thus ensure autophagosome elongation.
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1.6.2.2 Lipid trafficking at ER-mitochondria contact sites
Mitochondria are ancient eukaryotic endomembranes that are considered as the “energy powerhouse” of eukaryotic cells 147148. In mammals, around 3-10 % of mitochondrial surface is associated with the ER depending on the cell type 87. The physiological role of ER- mitochondria contact sites was first thought to be limited to lipid and calcium exchange between both organelles 102149150. However, it is becoming more and more evident that ER- mitochondria contact sites also play an essential role during organelle remodeling events such as mitochondrial fission 91 151. Interestingly, dysfunctional contacts between ER and mitochondria have been implicated in multiple human pathologies 152153154155156.
Mitochondria integrity entirely depends on its communication with the ER since they obtain most of its lipids directly from ER membranes 157158159160. Nevertheless, some phospholipids such as cardiolipin (CL), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) can be synthesized at the inner mitochondrial membrane (IMM) 161 162 163164. In any case, all these phospholipids require phosphatidic acid (PA) as a precursor, which is also imported from the ER 55 101. Lipid exchange between both organelles occurs at specific ER subdomains called mitochondria-associated membranes (MAMs), where phospholipid synthesizing enzymes accumulate 165166. A classic example of how lipids traffic between both membranes is depicted in Figure 9. Interestingly, yeast mutants that can only make phosphatidylethanolamine (PE) and phosphatidylcholine (PC) by this pathway grow normal and have similar levels of PE and PC as the WT strains 167. This data indicates that non-vesicular trafficking between ER and mitochondria is highly efficient 118.
Figure 9 l Lipid transport at ER-mitochondria contact sites. PA (phosphatidic acid) is converted to PS (phosphatidylserine) in the ER and transferred to the inner mitochondrial membrane (MM) where is converted to PE (phosphatidylethanolamine). PE travel back to the ER and is converted to PC (phosphatidylcholine), which is likely to be shuttle also back to the mitochondria. The identity of the proteins responsible of the lipid transport is unknown. However, some molecular tethers have been identified in both yeast (ERMES and EMC complex) and mammalian cells (EMC complex and multiple paired tethers).
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The identity of LTPs at ER-mitochondria contacts sites remains elusive. Only recently has been reported that the LTPs ORP5/8 (Oxysterol-binding protein-related protein 5/8) are able to localize at ER-mitochondria contacts 168. Strikingly, it has been demonstrated that phospholipid transport between ER and mitochondria doesn´t require cytosolic factors suggesting that LTPs are not strictly necessary for lipid trafficking at MAMs 118169. Therefore, one possibility is that lipid trafficking occurs through the formation of hydrophobic channels between both membranes 133 135 170 171. Additionally, transient hemifusion of ER and mitochondrial membranes may also facilitate lipid exchange independently of LTPs.
In this way, maintain both ER and mitochondrial membranes closely appose each other is needed for sustaining proper lipid trafficking between both organelles. In yeast, the ERMES complex has been proposed as a firm candidate for tethering the non-vesicular transport of phospholipids at ER-mitochondria contact sites 172 173 174. ERMES depletion decreases ER- mitochondria contact sites and slow down the metabolic flow of phospholipids between both membranes 172 175 176 177. Interestingly, some components of ERMES complex contain SMP domains that are able to bind lipids and form hydrophobic channels between different membranes 134178179 (Figure 9). However, it is unknown if ERMES components transfer lipids itself or are only structural proteins that tether ER and mitochondria to facilitate lipid transport. Surprisingly, phospholipids are still able to reach mitochondrial membranes in ERMES mutants through the vCLAMP (vacuole and mitochondria patch) complex 180. In fact, depletion of ERMES complex leads to an increase in vCLAMP in order to compensate the deficient phospholipid transport toward the mitochondria 181 182. Accordingly, depletion of both vCLAMP and ERMES is lethal demonstrating that both MCS cannot be simultaneously disturbed 182. Recent studies have also identified the EMC (ER membrane protein complex ) family of proteins as another tethering complex in both yeast and mammals 183184. In line with this, it has been demonstrated that EMC proteins directly interact with the TOM complex on mitochondrial membranes to mediate the tethering 183 (Figure 9). Consistently, EMC mutants show reduced ER-mitochondrial contacts and altered mitochondrial phospholipid composition.
As expected, double mutants for both EMC and ERMES are not viable 183.
Until 2017, orthologs of ERMES had been only found in yeast. However, Hirabayashi and colleagues have recently identified the protein PDZD8 (PDZ domain-containing protein 8) as a MAM resident protein in mammals that harbors a SMP domain functionally related to the ERMES component Mmm1 185. In this connection, PDZD8 resulted to be necessary for the formation of ER-mitochondria contact sites 185. Additionally, multiple tethering complexes between ER and mitochondria have been reported during the last years in mammalian cells,
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although it is unknown if they are required for lipid trafficking. These include Ins₃PR (Inositol trisphosphate receptor) and VDAC (Voltage-dependent anion chanel) 186, VAPs and PTPIP51 (Protein tyrosine phosphatase interacting protein 51) 187, BCAP31 (B-cell receptor-associated protein 31) and FIS1 (Fission 1 homologue) 188 and homoligomers of MFN2 (Mitofusin-2) 189 (Figure 9).
1.6.2.3 Lipid trafficking at ER-endosome contact sites
Endosomes entail a heterogeneous compendium of different endomembranes that participate in the sorting of internalized material from the plasma membrane to both lysosomes and Golgi apparatus 190191. Once early endosomes begin to mature, they are increasingly guided by interactions with the ER. It has been reported that around 50% of Early Endosomes (EE) and 99% of Late Endosomes (LE) are in contact with the ER as they traffic 96. ER-endosome contact sites have two main functions: 1) control endosome positioning 192 193 and 2) regulate lipid trafficking between both organelles 194. These two functions are related each other since lipid composition of endosomal membranes determines its position in the cell 194. Additionally, ER tubules have been also involved in endosome fission 97.
Cholesterol trafficking has been the most characterized lipid transport between ER and endosomes 194195196197. Cholesterol enters the cells through the endocytosis of LDL, which are cholesterol-enriched particles 198. Once inside the cell, at least 30% of this cholesterol is directly transferred to the ER 199. In this way, the first step is the dissociation of LDL from its receptor and the formation of unesterefied cholesterol by the hydrolytic action of lipases in the lumen of late endosomes (LE) 200. Then, the soluble LTP NPC2 (Niemann-Pick disease type C2) delivers cholesterol to NPC1 (Niemann-Pick disease type C1) 201, which is an integral membrane protein of LE and lysosomes that transfers sterols to ER membranes 202. It has been reported that NPC1 interacts with the ER-resident protein ORP5 at ER-endosome contact sites to accomplish cholesterol trafficking via the cholesterol-binding domain of ORP5 203204 (Figure 10A). Accordingly with this model, depletion of either NPC1 or ORP5 leads to the accumulation of sterols on late endosomal membranes 203. The reverse transport of cholesterol from the ER to endosomes is less documented. Recent reports have demonstrated that the LTPs ORP1L (Oxysterol-Binding Protein-Related Protein 1L) and STARD3 (StAR-related lipid-transfer 3) transport cholesterol to endosomal membranes via its interaction with VAPs proteins 92205206 (Figure 10A). Interestingly, both ORP1L and cholesterol levels on endosomal membranes determine endosome positioning in the cell 207208209. In this fashion, when cholesterol is high on endosomal membranes, dynein associates with endosomes to promote their transport to
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the juxtanuclear zone. However, under low cholesterol conditions on endosomes, ORP1L interacts with the ER protein VAPA (Figure 10A) , resulting in the release of the dynein motor, and thus halting the transport toward lysosomes 207209210.
ORP/OSBP family of proteins are also able to exchange other lipids such as PtdIns4P at MCS 211
212. In the model proposed by Dong et al .211 (Figure 10B), VAPs serve as pivotal platforms that stabilize ER-endosome contacts by interacting with both SNX2 on endosomal membranes and the cytosolic LTP OSBP (Oxysterol Binding Protein). OSBP transfers PtdIns4P from endosomes to the ER, where it is rapidly hydrolyzed by the PtdIns4P phosphatase SAC1 in order to maintain lipid gradients and avoid the return of PtdIns4P to endosomal membranes.
Consistently, absence of either VAPs, OSBP or SAC1 results in abnormal accumulation of PtdIns4P on endosomes and compromised endosomal function 211. Particularly, PtdIns4P trafficking at ER-endosome contact sites has been studied in this thesis and thus will be further discussed in both Results and Discussion sections.
1.6.2.4 Lipid trafficking at ER-Golgi contact sites
The Golgi complex serves a multitude of functions including protein secretion and modification
213214. Communication between Cis-Golgi and ER has been largely reported during the study of the vesicular secretory pathway 215 216 217. Nevertheless, the ER also encounters with trans- Golgi (TGN) cisternae at MCS 218219.
In fact, one of the most convincing evidences of non-vesicular trafficking between different organelles is supported by studies at ER-TGN contact sites. CERT (Ceramide Transfer Protein)
Figure 10 l Lipid transport at ER-endosome contact sites. (A) Several complexes are involved in control cholesterol trafficking between ER and endosomes. For example, NPC1 (LE) and ORP5 (ER) cooperate to transfer cholesterol from LE to the ER. STARD3 and ORP1L interact with VAPs to transport cholesterol in the opposite direction when endosomal cholesterol levels are low. (B) PtdIns4P trafficking also occurs at ER-endosome contacts. OSBP binds to both SNX2 on endosomes and VAPs at the ER to facilitate the delivery of PtdIns4P from endosomes to the ER resident phosphatase SAC1. Figure B is based and modified from Dong et al. 211.
