Anusara Daenthanasanmak ZIB Jason Mercer and Ari Helenius Nature Cell Biology volume 11 May 2009

1. Endocytosis 2. Macropinocytosis 3. Cellular factors and signalling pathways 4. Viruses that internalized via macropinosomes 5. Perspectives

Nature of the cargo Cellular factors Signal needed for activation The fate of internalised material

Actin dependent endocytosis Internalization of fluid and membrane Plasma membrane ruffling

Inhomogenous in size and irregular in shape Diameter of µm Increase cellular fluid 5-10 fold

Role of particle Fluid uptake Cargo specificity Cell-type specificity Cellular machinery Global versus membrane activation

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

Na+/H+ exchangers The inhibitors are sometimes used as the diagnostic test to identify Macropinocytosis Depletion of cholesterol blocks both membrane ruffling and macropinocytosis

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

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

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

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

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

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 α v -integrins triggering several cellular responses including activation of PI(3)K The virus activates Pak1 and CtBP1 for closure of Ad3-macropinosome

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

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

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

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

HIV-1 virions enter large uncoated intracellular vacuoles into Macrophages (Marechal et al. J.virol 2001)

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

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

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