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Anusara Daenthanasanmak
ZIB 15.11.2010
Jason Mercer and Ari Helenius
Nature Cell Biology volume 11May 2009
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1. Endocytosis
2. Macropinocytosis
3. Cellular factors and signalling pathways
4. Viruses that internalized via macropinosomes
5. Perspectives
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• Nature of the cargo
• Cellular factors
• Signal needed for activation
• The fate of internalised material
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• Actin dependent endocytosis
• Internalization of fluid and membrane
• Plasma membrane ruffling
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• Inhomogenous in size and irregular in shape
• Diameter of 0.5-10 µm
• Increase cellular fluid 5-10 fold
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• Role of particle
• Fluid uptake
• Cargo specificity
• Cell-type specificity
• Cellular machinery
• Global versus membrane activation
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• A family of hydrolase enzymes that can bind and hydrolyze guanosine triphosphate (GTP)
• Small GTPases regulate a wide variety of processes in the cell, including growth, cellular differentiation, cell movement and lipid vesicle transport
• Ras Superfamily GTPase play a crucial role
• Ras superfamily is divided into eight main families Ras, Rad, Rab, Rap, Ran, Rho, Rheb, Rit, and Arf
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• Na+/H+ exchangers
• The inhibitors are sometimes used as the diagnostic test to identify Macropinocytosis
• Depletion of cholesterol blocks both membrane ruffling and macropinocytosis
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• Macropinosomes are sensitive to cytoplasmic pH
• In human carcinoma A431 cells, most macropinosome recycle back to the cell surface
• Trafficking seems to depend on cell type and mode of induction
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• Type of endocytosis used is determined by particle, size, choice of receptor, cell tropism and mode of transmission
• Viruses are valuable tools for the study of endocytic mechanisms
• Viruses can make use of Macropinocytosis; directly or indirectly
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1. Ruffling
2. Fluid uptake
3. Actin
4. Rho GTPases
5. Na/H exchangers
6. Kinases – inhibitors of Pak1, PI(3)K and PKC kinases block entry
7. Other factors – dynamin-2, myosin II, microtubules and Arf6
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• Poxvirus family, replicate in cytosol
• Large, enveloped, double stranded DNA virus
• Mature Vaccinia virions mimic the uptake of apoptotic bodies and enter cells using macropinocytosis
• The uptake causes a rapid, transient increase of fluid phase markers
• Inhibitor analysis shows that Pak1, PKC and PI(3)K are also needed
• Na/H exchangers, myosin II and cholesterol are required
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d. Focused ion beam-scanning electron microscopy (FIB-SEM): virions internalized next to retracting blebs
C. Vaccinia virus mature virions induces systemic blebbing in HeLa cells
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• Non-enveloped, double stranded DNA viruses
• Human adenovirus serotype 3 (Ad3) is associated with epidemic conjunctivitis, fatal respiratory and systemic disease
• Its entry into epithelial and haematopoietic cells by direct macropinocytosis by binding to CD46 activates Rac1
• Ad3 induces clustering of αv-integrins triggering several cellular responses including activation of PI(3)K
• The virus activates Pak1 and CtBP1 for closure of Ad3-macropinosome
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• Picornaviridae family
• Small, non enveloped RNA virus
• Causing meningoencephalitis, carditis and mild respiratory or enteric disease
• EV1 bind α2β1-integrins and co-internalised into vacuolar that accumulate fluid-phase markers
• Infection is dependent on actin dynamics and Rac1
• It also requires cholesterol, Pak1, PI(3)K, PLC and Na/H exchanger
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• Picornavirus
• CVB can cause myocarditis and hepatitis
• Enter epithelia at tight junctions by stimulating the internalization of junctional membrane and virus itself into macropinosome
• Internalized virions colocalize with fluid phase makers and the Rab5 effector
• CVB entry requires the activity of Ras, Rab5 and Rab34 GTPase, Na/H exchangers and PKC
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• Large enveloped double stranded DNA virus
• Cause mucosal blisters to deadly brain infections
• HSV1 shows cell type dependent entry mechanisms
• Macropinocytosis occurs in epidermal keratinocytes, HeLa and CHO cells
• EM shows HSV1 virions to be in large, uncoated vesicles during the early stage of internalization
•Inhibition of PI(3)K or RTKs prevent virus entry and infection
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• Enveloped, single stranded RNA lentivirus
• HIV-1 are internalized by cell type-specific entry mechanisms
• Marechal et al. (2001) showed direct Macropinocytosis in human macrophages and Brain microvascular endothelial cells (BMVECs) (Liu et al, 2002)
• EM of HIV-1 infected BMVECs showed internalization into large cytoplasmic vacuoles with fluid phase makers
• Macropinocytosis of HIV-1 into BMVECs is not a productive infection route, but rather for cell-to- cell transmission
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HIV-1 virions enter large uncoated intracellular vacuoles into Macrophages (Marechal et al. J.virol 2001)
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• Associate with respiratory infections
• Ad2/5 binds to its receptor induces the integrin clustering needed for internalization
•Ad2/5-induced macropinosomes are lysed and their contents released into the cytosol, required for escaping and infection
• Ad2/5 triggered macropinocytosis depends on integrins, PKC, actin dynamics, Rac1, Na/H exchange and cholesterol, independent of dynamin
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• Small, enveloped single-stranded RNA
• Cause rash and fever
• EM showed RV within clathrin-coated pits and vesicles and particles also colocalized with clathrin-mediated makers
• RV enters cells by clathrin-mediated endocytosis, however, inhibition of Na/H exchange, actin or microtubule polymerization inhibits macropinocytosis
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• Are there several different types of macropinocytosis?
• How many different viruses are using these pathways?
• How do they trigger the relevant responses in different host cell types?
• Why a virus might use macropinocytosis for immune evasion?
• Macropinocytosis of apoptotic debris is known to suppress activation of innate immune responses
• Ruffling and increased motility, in vivo infection, may allow viruses to spread more efficiently through epithelial layers, basal lamina and other obstacles
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