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Dr. Jafar Rezaie  / Assistan Professor

Contact information: Mail: Rezaie.j@umsu.ac.ir  Tell:+989148548503, Fax:+984432240642

 

 

I am Jafar Rezaie who is majoring in Molecular and Stem Cell Biology. I am working in field of Extracellular Vesicles (EVs)  biology especially  focused on exosome signaling pathway in cancer. EVs contribute to physiological and pathological processes in a specific manner. In the field of cance biology, scientists have interested in studying EVs biology and as well in the application of EVs in diagnosis and treatment of cancers. Here, I am interested in lightening the key role of EVs in cancerous tissues In Vivo/In Vitro, and also following their bio-application in order to design effective delivery system and introduce specific exosome as biomarkers in cancer disciplines.

 

 

Research Focus


  1.  Exosome signaling pathway  in cancer.
  2.  Use Exosomes as biomarkers.
  3. Exosome based drug delivery systems.

 

:Team Work Group members

      Dr. N.Jabbari, PhD

  Dr. H.Soraya, PhD

 

?What are exosomes

12:01:32  دوشنبه ٢٦ شهريور ١٣٩٧

In the past decade, EVs are known to mediate intercellular communication in various cell types. EVs represent heterogeneous populations of very small, bi-phospholipid membranous particles of cells with ability to carry bioactive molecules such as protein, lipid, mRNA, miRNA. Even, it was proved that small DNA sequences are also released by almost eukaryotic cells. Three major subpopulations of EVs are generally identified based on the origin, way of generation and size: (i) exosomes, (ii) MVs, and (iii) apoptotic bodies (ABs). Exosomes belong to a large family of membrane vesicles referred to as extracellular vesicles. First, exosomes were discovered in 1983 by two independent work groups directed by Harding and Pan. In 1983, Johnstone et al. termed these vesicles as “exosomes”. Exosomes, nano-sized (30-120 nm) membranous vesicles, are directly originated via intercellular regulated process. These small particles, generated from endosomal compartment, multivesicular body (MVB), are routinely located in the cytoplasm. It is clear that the invagination of MVB membrane results in the formation of intraluminal vesicles (ILVs) in MVBs lumen. MVBs could selectively fuse with a lysosome to degrade their contents. Alternatively, MVBs may fuse with the plasma membrane and release ILVs contents to extracellular space as exosome for the purposes of intercellular transport and signaling. Scientists believe that MVB membrane composition determines the fate of MVBs. For abscission of MVs, Rab-GTPase family mediates intracellular trafficking and docking of MVB to plasma membrane . Most experimental evidence suggests that many molecules contribute to exosome biogenesis and cargo sorting. For instance,  endosomal sorting complex required for transport (ESCRT) machine is complexes of diverse proteins that located on the outer side of the MVB membrane are involved in the sorting of ubiquitinated proteins into exosomes. In the ESCRT independent mechanism, other tetraspanins such as CD63, CD81, and CD82, abundantly founded on exosome membrane, mediate exosome biogenesis. Observations confirmed the involvement of sphingomyelinase, protein kinase D family, trinucleotide repeat-containing gene 6A protein (TNRC6A), and Argonaute-2 (AGO2) in exosome biogenesis and cargo packagin. The common molecular markers are tetraspanins, CD9, CD63, CD81, CD82, CD151, intercellular adhesion molecule-1 (ICAM1), CD51, CD61, Alix, milk fat globule-E8/lactoferrin, CD80, CD86, CD96, Rab5b, and major histocompatibility complex (MHC) class, I and II. Various cells including non-stem/stem cells have capability to release exosomes into the surrounding milieu. These nanovesicles have both favorable physiological rules and even harmful pathological roles in biological processes. Using transmission electron microscopy analysis, exosomes are represented as cup-shaped vesicles with a diameter between ∼30–120 nm, but the spherical appearance of exosomes will be achieved when cryoEM technique is used, showing the real-state exosomes morphology.

 

                                              Exosome Biogenesis, Secretion and Uptake :

 

 

: Exosomes Biogenesis and their fate

 

 

 

 

Provided by Dr. J.Rezaie

 

 

 

 

دوشنبه ٢ مهر ١٣٩٧      16:08:31  

 

 Exosome biogenesis

          The current idea in field of exosome is that the endocytic pathway was initially thought of as a mechanism to produce exosomes. Clathrin-dependent endocytosis involves the formation of clathrin-coated pits along the plasma membrane (PM), which causes inward budding of the membrane into intracellular vacuole named early endosom, which undergoes several changes as it matures to form a late endosome (9). Late endosomes, because of their specific feature then also called multivesicular body (MVB) have a round shape, representative their maturation from the vacuole domain of the early endosomes (10). The surrounding membrane of the late endosome contains several molecules for example lipid rafts (cholesterol and spingolipid rafts), tetraspanins (CD9, CD63, CD81), clathrin, and components of ESCRT machinery (3, 8). The first intracellular step in exosome biogenesis involves the invagination of the plasma membrane of multivesicular body (MVB) which form a membrane bound vesicles in MVBs lumens that identified as intraluminal vesicles (ILVs) (Fig. 1). Many molecules have been considered in biogenesis, secretion of exosmes in regular manner(11). Recently, numerous studies have indicated that the ESCRT machinery selectively contribute to form and pack exosome cargoes in corporation with different accessory proteins (Fig.1)(2). ESCRT machinary composed of four complexes, ESCRT-0, ESCRT-I ESCRT-II ESCRT-III and also accessory proteins. ESCRT-0 is the initial complex attributed in ILVs biogenesis and target proteins on cytosolic side of MVBs. In this context, ESCRT machinery selectively captured the monoubiquitinated proteins.  The interaction of the ESCRT- 0 complex subunit, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), with the lipid phosphatidylinositol 3-phosphate, located on the MVB membrane, ignite the ESCRT machinery function and binds HRS to ubiquitylated proteins. Then, ESCRT-0 recruits the ESCRT-I component, resulting in combining of ESCRT-II subunits. The connection of ESCRT-I and - II recruits the reverse budding of the MVB membrane. Inside the border of the emerging ILVs, the ESCRT-III complex joined to ESCRT-II, inducing the nascent vesicle abscission. In final step, using an ATPase, the ubiquitin tag and the ESCRT subunits are disassembled from MVB membrane, but some ESCRT components and accessory proteins such as TSG101, HRS, and ALG2-interacting protein X (ALIX)] are associated with the secreted exosomes  (Fig. 2) (11, 12). More interestingly, studies in some mammalian cell lines have demonstrated the formation of MVBs in absent of the ESCRT machinery. It seems that in oligodendroglial cells ceramide is a key molecule to induce inward budding of the limiting membrane of late endosomes in absent of the ESCRT machinery (3, 13).  For example a work showed that an inhibition of neutral sphingomyelinase, the enzyme that catalyzes the biosynthesis of ceramide, resulted in less secretion of proteolipid (PLP) containing exosomes (13). The other work conducted by Kulshreshtha and colleague showed that Inhibition of neutral sphingomyelinase with GW 4869 successfully resulted in blocking exosomal release from epithelial cells in the asthmatic model of mouse (14). In addition, phosphatidic acid has also been shown to contribute inward budding of the limiting membrane of late endosomes to induce ILV production (15, 16). The ESCRT-dependent and ceramide (ESCRT-independent) sorting mechanisms have been shown in (Fig. 2).  Based on such observations, while the ESCRT machinery requires monoubiquitination of cargoes, some cargos were sorted by independent of ubiquitin tags. For example, melanosomal protein PMEL  undergoes sorting in an ubiquitin-independent manner to clathrin subdomains of MVBs which the depletion of Hrs, Tsg101, and Vps4 in melanoma cells did not diminished it sorting (17).  Similarly, RNAs such as miRNAs are sorted into MVBs in ubiquitin-independent mechanisms. Since RNP complexes possess RNA binding motifs were founded in exosomes, it is dialectician to propose that the ribonucleoprotein (RNP) complexes are attributed in sorting miRNAs and other noncoding RNA molecules (18). To confirmation, Villarroya-Beltri et al. discovered that miRNAs bearing specific sequence motifs that transfer them to the protein heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1), which loads miRNAs into ILVs (19). As fact, it is not clear that whether cytoplasmic and other membrane-originated molecules were sorted through possible unknown mechanisms or were loaded into in randomized manner.  After sorting of cargo (proteins and RNAs) into ILVs, the late endosome converts into a fully matured which named MVB (20).

3.2.3 Signaling in Intracellular traffic and fate of MVBs

Intracellular trafficking of vesicle systems was conducted by RabGTPase family proteins. In human cells more than 60 Rab proteins were characterized which preferentially associated with intracellular compartment (2). As shown in Fig several Rab proteins are specifically contribute to transfer of vesicles in definitive pathways.  For example, molecular insight into subcellular signaling revealed the association of Rab-31 with vesicles which come from Golgi apparatus.  The process for intracellular trafficking of MVBs to the cell periphery and the fusion of MVBs with the cell membrane involves recruitment of Rab-11, Rab-27a, Rab-27b, and Rab-35 proteins on limiting membrane of definitive MVBs. Moreover, it is suggested that Rab-4, Rab-7 were utilized in signaling other alternative MVBs pathways (2, 8, 11). The finding that Knock down of expression of Rab-2B, Rab-9A, and Rab-5A inhibits exosome secretion in tumor cells indicated involvement of these Rab proteins in cancer cells (21, 22). It appears clear that, the heterogeneity in the necessity for docking and fusion machineries could propose the co-existence of diverse MVBs within the cell. Again, in various cells the involvement of same Rab proteins in common pathways is under dubiety and needs to elucidate preference and specialty of Rab proteins in trafficking intracellular pathways. In addition, the involvement of soluble NSF attachment protein receptor (SNARE) proteins has been suggested to control the fusion and of MVBs with the plasma membrane (20). For example, RNAi silencing of Ykt6, a SNARE protein, is resulting in intracellular accumulation of exosomal marker GFP-CD63 in the cell (23). Fader and et al. revealed that VAMP-7, a SNARE,  regulates MVBs fusion with PM to release acetylcholinesterase-loading EVs in K562 cell line (24). Such observations were confirmed the association of some SNARE proteins such as SNAP-23, VAMP-7, and VAMP-8 in the Ca2+-regulated fusion of secretory lysosomes with the PM in various cell types (25-27)Reversely, the inhibition of VAMP-7 in Madin-Darby canine kidney cells impaired lysosomal secretion, but not secretion of HSP70-bearing EVs (28). However, the precise mechanism underlying the fusion and release of MVBs with PM remains largely unknown. Three different fates have been identified for the fully matured MVB (2) (Fig.). MVBs could selectively fuse with a lysosome to degrade their contents in order to dampen ligand-induced signals. Alternatively, MVBs may fuse with the PM and release ILVs contents to ECM as exosome for the purposes of intercellular transport and signaling. In addition, MVBs may directly merge to PM to present specific molecules. It is suggested that the lipid composition ratio of MVBs surrounding membrane is landmark to determine that MVB could be degrade or evacuate its cargos (5)

3.2.4 Mechanisms of Exosomes Uptake

 

          Once secreted, exosomes receive to neighboring cells or to the extracellular matrix, or passively traffic through the bloodstream or through other bio-fluids to target recipient cells. Increasing evidence suggests that three possible mechanisms may be involved in  exosome uptake; (i) internalization; (ii) direct fusion; (iii)  receptor-ligand interaction (Fig) (29, 30). In the internalization pathway, exosome were taken up by cells via a variety of endocytic pathways, including clathrin-dependent endocytosis, caveolin-coated endocytosis, macropinocytosis, phagocytosis, and lipid raft-mediated internalization (31). The second mechanism is direct fusion, in which membrane actively fuse and then exosome cargo was injected into the cytoplasmic  unit of recipient cells (32). In this pathway , membrane fusion is mediated by several proteins includin Rab GTPase proteins, SNAREs, and Sec1/Munc-18 related proteins (SM-proteins) (33). Finally, exosome could initiate cell signaling by using ligand-receptor interaction, in which adhesion molecules located on exosome surface membrane connect directly or indirectly to itself ligands present on target cell membrane. For example, mature DCs derived exosomes bearing on their surface the intercellular adhesion molecule 1 (ICAM1) that bind to the lymphocyte function-associated antigen 1 receptor(LFA1-R) localized on the PM of antigen presenting cells (APCs) (34) or stimulated T-cells (35). In exosome-based delivery systems, it is biologically valuable to understand the underling mechanisms in exosome uptake that engineer effective and specific exosomes and discovery of intercellular signaling.  

 

 

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