Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

10.32920/14638422.v1 ◽

2021 ◽

Author(s):

Farnaz Fekri ◽

Ralph Christian Delos Santos ◽

Raffi Karshafian ◽

Costin N. Antonescu

Keyword(s):

Drug Delivery ◽

Fluid Phase ◽

Acid Sphingomyelinase ◽

Coated Pits ◽

Drug Permeability ◽

Membrane Pores ◽

Signaling Mechanisms ◽

Sheer Stress ◽

Cell Plasma ◽

Fluid Phase Endocytosis

Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted drug delivery.

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Structure-based inhibitors reveal roles for the clathrin terminal domain and its W-box binding site in CME

10.1101/2020.01.24.919092 ◽

2020 ◽

Author(s):

Zhiming Chen ◽

Rosa Mino ◽

Marcel Mettlen ◽

Peter Michaely ◽

Madhura Bhave ◽

...

Keyword(s):

Binding Sites ◽

Fluid Phase ◽

Structure Design ◽

Coated Pits ◽

Bulk Fluid ◽

Lysosomal Trafficking ◽

Terminal Domain ◽

Binding Surface ◽

Fluid Phase Endocytosis ◽

Late Stages

AbstractClathrin-mediated endocytosis (CME) occurs via the formation of clathrin-coated vesicles from clathrin-coated pits (CCPs). Clathrin is recruited to CCPs through interactions between the AP2 complex and its N-terminal domain (TD), which in turn recruits endocytic accessory proteins. Inhibitors of CME that interfere with clathrin function have been described, but their specificity and mechanisms of action are unclear. Here we show that overexpression of the TD with or without the distal leg specifically inhibits CME and CCP dynamics by perturbing clathrin interactions with AP2 and SNX9. We designed small membrane-penetrating peptides that mimic the four known binding sites on the TD. A peptide, Wbox2, designed to mimic to the W-box motif binding surface on TD binds to SNX9 and AP2, and potently and acutely inhibits CME, while not perturbing AP1-dependent lysosomal trafficking from the Golgi or bulk, fluid phase endocytosis.SummaryChen et al define the role the N-terminal domain (TD) of clathrin heavy chain in early and late stages of clathrin-mediated endocytosis, and guided by its structure, design a membrane-penetrating peptide, Wbox2, that acutely and potently inhibits CME.

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The protein tyrosine kinase p56lck inhibits CD4 endocytosis by preventing entry of CD4 into coated pits

The Journal of Cell Biology ◽

10.1083/jcb.117.2.279 ◽

1992 ◽

Vol 117 (2) ◽

pp. 279-290 ◽

Cited By ~ 92

Author(s):

A Pelchen-Matthews ◽

I Boulet ◽

DR Littman ◽

R Fagard ◽

M Marsh

Keyword(s):

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Fluid Phase ◽

Endocytic Pathway ◽

Coated Pits ◽

Protein Tyrosine ◽

Src Gene ◽

Fluid Phase Endocytosis ◽

Nih 3T3

The lymphocyte glycoprotein CD4 is constitutively internalized and recycled in nonlymphoid cells, but is excluded from the endocytic pathway in lymphocytic cells (Pelchen-Matthews, A., J. E. Armes, G. Griffiths, and M. Marsh. 1991. J. Exp. Med. 173: 575-587). Inhibition of CD4 endocytosis is dependent on CD4 expressing an intact cytoplasmic domain and is only observed in cells where CD4 can interact with the protein tyrosine kinase p56lck, a member of the src gene family. We have expressed p56lck, p60c-src, or chimeras of the two proteins in CD4-transfected NIH-3T3 or HeLa cells. Immunoprecipitation of CD4 and in vitro kinase assays showed that p56lck and the lck/src chimera, which contains the NH2 terminus of p56lck, can associate with CD4. In contrast, p60c-src and the src/lck chimera, which has the NH2 terminus of p60c-src, do not associate with CD4. Endocytosis assays using radioiodinated anti-CD4 monoclonal antibodies demonstrated that coexpression of CD4 with p56lck, but not with p60c-src, inhibited CD4 endocytosis, and that the extent of the inhibition depended directly on the relative levels of CD4 and p56lck expressed. The uptake of mutant CD4 molecules which cannot interact with p56lck was not affected. Measurement of the fluid-phase endocytosis of HRP or the internalization of transferrin indicated that the effect of p56lck was specific for CD4, and did not extend to other receptor-mediated or fluid-phase endocytic processes. Immunogold labeling of CD4 at the cell surface and observation by electron microscopy demonstrated directly that p56lck inhibits CD4 endocytosis by preventing its entry into coated pits.

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ICAM-1 recycling in endothelial cells: a novel pathway for sustained intracellular delivery and prolonged effects of drugs

Blood ◽

10.1182/blood-2004-05-1714 ◽

2005 ◽

Vol 105 (2) ◽

pp. 650-658 ◽

Cited By ~ 86

Author(s):

Silvia Muro ◽

Christine Gajewski ◽

Michael Koval ◽

Vladimir R. Muzykantov

Keyword(s):

Drug Delivery ◽

Endothelial Cells ◽

Fluid Phase ◽

Therapeutic Agents ◽

Intercellular Adhesion Molecule ◽

Major Fraction ◽

Intercellular Adhesion Molecule 1 ◽

Lysosomal Trafficking ◽

Fluid Phase Endocytosis

AbstractIntercellular adhesion molecule-1 (ICAM-1) is a target for drug delivery to endothelial cells (ECs), which internalize multivalent anti-ICAM nanocarriers (anti-ICAM/NCs) within 15 to 30 minutes. The concomitant ICAM-1 disappearance from the EC surface transiently inhibited subsequent binding and uptake of anti-ICAM/NCs. Within 1 hour, internalized ICAM-1 diverged from anti-ICAM/NCs into prelysosomal vesicles, resurfaced, and enabled uptake of a subsequent anti-ICAM/NC dose. Thus, internalized ICAM-1 was able to recycle back to the plasma membrane. In vivo pulmonary targeting of a second anti-ICAM/NC dose injected 15 minutes after the first dose was decreased by 50% but recovered between 30 minutes and 2.5 hours, comparable to cultured ECs. Anti-ICAM/NCs affected neither EC viability nor fluid-phase endocytosis and traffic to lysosomes. However, lysosomal trafficking of the second dose of anti-ICAM/NCs was decelerated at least 2-fold versus the first dose; hence the major fraction of anti-ICAM/NCs resided in prelysosomal vesicles for at least 5 hours without degradation. Two successive doses of anti-ICAM/NC/catalase protected ECs against H2O2 for at least 8 hours versus 2 hours afforded by a single dose, suggesting that recurrent targeting to ICAM-1 affords longer effects. ICAM-1 recycling and inhibited lysosomal traffic/degradation of subsequent doses may help to prolong activity of therapeutic agents delivered into ECs by anti-ICAM/NCs.

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Molecular Mobility in Trehalose Loaded Mammalian Cells: Time-Resolved Fluorescence Anisotropy Measurements

ASME 2008 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2008-193077 ◽

2008 ◽

Author(s):

Nilay Chakraborty ◽

Wesley Parker ◽

Kevin E. Elliott ◽

Stuart T. Smith ◽

Patrick J. Moyer ◽

...

Keyword(s):

Mammalian Cells ◽

Fluid Phase ◽

Water Soluble ◽

Great Promise ◽

Time Resolved ◽

Intracellular Space ◽

Membrane Pores ◽

Fluid Phase Endocytosis ◽

Membrane Bound ◽

Cellular Compartments

Many preservation methods have utilized sugars such as trehalose as protectants against injury during cell preservation processing, especially during drying (1–5). As mammalian cells do not synthesize trehalose, research in the mammalian cell desiccation field has focused on the development of strategies to enable trehalose delivery into the intracellular milieu. Numerous techniques have been explored ranging from microinjection (2) to the creation or utilization of membrane pores (1,3). Fluid phase endocytosis has shown great promise as an effective strategy for non-invasively delivering water-soluble materials into the intracellular space (4, 5). In this technique trehalose is transported across the cell membrane in membrane-bound cellular compartments called endosomes. Cells incubated in cell culture medium containing trehalose have been shown to take up considerable amounts of trehalose by this technique (4, 5). How much of this trehalose actually become available for protection of biomolecules during the dehydration process has yet to be determined.

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Phorbol ester-induced downregulation of CD4 is a multistep process involving dissociation from p56lck, increased association with clathrin-coated pits, and altered endosomal sorting.

Journal of Experimental Medicine ◽

10.1084/jem.178.4.1209 ◽

1993 ◽

Vol 178 (4) ◽

pp. 1209-1222 ◽

Cited By ~ 94

Author(s):

A Pelchen-Matthews ◽

I J Parsons ◽

M Marsh

Keyword(s):

Phorbol Ester ◽

Protein Tyrosine Kinase ◽

Phorbol Esters ◽

Fluid Phase ◽

Immunofluorescent Staining ◽

Coated Pits ◽

Endosomal Sorting ◽

Fluid Phase Endocytosis ◽

Multistep Process ◽

Nih 3T3

The phorbol ester phorbol myristate acetate (PMA) induces a rapid downregulation of CD4 from the surface of T cells and lymphocytic cell lines, as well as from CD4-transfected nonlymphoid cells. Here we have studied the mechanisms of this phorbol ester-induced CD4 modulation. Using HeLa-CD4 or NIH-3T3-CD4 cells, in which the endocytosis of CD4 is not influenced by the protein tyrosine kinase p56lck, we show that PMA enhanced the uptake of CD4, increasing the rate of CD4 endocytosis three to five-fold, and doubling the proportion of CD4 found inside the cells. Trafficking of a CD4 mutant lacking the major portion of the cytoplasmic domain, as well as fluid phase endocytosis were not affected by PMA treatment. Studies in which clathrin-coated pits were disrupted through the use of hypertonic media indicated that both the constitutive and PMA-induced CD4 uptake occurred through coated vesicles. Electron microscopy demonstrated directly that PMA increases the association of CD4 with coated pits. Immunofluorescent staining of internalized CD4 showed that PMA also diverted CD4 from the early endosome-plasma membrane recycling pathway to a mannose 6-phosphate receptor-containing late endosomal compartment. In lymphoid or p56lck-expressing transfected cells, these effects were preceded by the PMA-induced dissociation of CD4 and p56lck, which released CD4 and made possible increased endocytosis and altered intracellular trafficking. Together these results indicate that phorbol esters have multiple effects on the normal endocytosis and trafficking of CD4, and suggest that phosphorylation may influence the interaction of CD4 with coated pits.

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Nanoscale Membrane Pores Follow Two Tension-Triggered Pathways to Membrane Rupture During Exocytosis, Drug Delivery and Transfection

Biophysical Journal ◽

10.1016/j.bpj.2020.11.2036 ◽

2021 ◽

Vol 120 (3) ◽

pp. 322a

Author(s):

Dong An ◽

Sathish Thiyagarajan ◽

Egor Antipov ◽

Brett E. Alcott ◽

Ben O'Shaughnessy

Keyword(s):

Drug Delivery ◽

Membrane Pores ◽

Membrane Rupture

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Synaptojanin family members are implicated in endocytic membrane traffic in yeast

Journal of Cell Science ◽

10.1242/jcs.111.22.3347 ◽

1998 ◽

Vol 111 (22) ◽

pp. 3347-3356 ◽

Cited By ~ 2

Author(s):

B. Singer-Kruger ◽

Y. Nemoto ◽

L. Daniell ◽

S. Ferro-Novick ◽

P. De Camilli

Keyword(s):

Synaptic Vesicles ◽

Direct Evidence ◽

Family Members ◽

Fluid Phase ◽

Growth Defect ◽

Temperature Sensitive ◽

Close Link ◽

Fluid Phase Endocytosis ◽

Synaptojanin 1

The synaptojanins represent a subfamily of inositol 5′-phosphatases that contain an NH2-terminal Sac1p homology domain. A nerve terminal-enriched synaptojanin, synaptojanin 1, was previously proposed to participate in the endocytosis of synaptic vesicles and actin function. The genome of Saccharomyces cerevisiae contains three synaptojanin-like genes (SJL1, SJL2 and SJL3), none of which is essential for growth. We report here that a yeast mutant lacking SJL1 and SJL2 (Deltasjl1 Deltasjl2) exhibits a severe defect in receptor-mediated and fluid-phase endocytosis. A less severe endocytic defect is present in a Deltasjl2 Deltasjl3 mutant, while endocytosis is normal in a Deltasjl1 Deltasjl3 mutant. None of the mutants are impaired in invertase secretion. The severity of the endocytic impairment of the sjl double mutants correlates with the severity of actin and polarity defects. Furthermore, the deletion of SJL1 suppresses the temperature-sensitive growth defect of sac6, a mutant in yeast fimbrin, supporting a role for synaptojanin family members in actin function. These findings provide a first direct evidence for a role of synaptojanin family members in endocytosis and provide further evidence for a close link between endocytosis and actin function.

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Effect of serum proteins on sodium-linked transport processes in cultured kidney cells.

Journal of the American Society of Nephrology ◽

10.1681/asn.v5111964 ◽

1995 ◽

Vol 5 (11) ◽

pp. 1964-1970

Author(s):

S S Blumenthal ◽

D L Lewand ◽

P A Tipnis ◽

J G Kleinman

Keyword(s):

Amino Acid ◽

Dextran Sulfate ◽

Cultured Cells ◽

Serum Proteins ◽

Fluid Phase ◽

Defined Medium ◽

Transport Processes ◽

Transport Systems ◽

Heat Treated ◽

Fluid Phase Endocytosis

The mechanism for increased Na+ retention in the nephrotic syndrome is unknown. To determine if Na+ transport systems in the proximal tubule might be affected by filtered proteins, mouse cortical tubule cells grown in defined medium were exposed to concentrations of bovine serum albumin (BSA) ranging from 0.01 to 0.5%. Activity of the Na(+)-glucose cotransporter, measured as Na(+)-dependent uptake of alpha-methylglucoside, increased progressively to a maximum of 2.3-fold above baseline (P < 0.001; N = 10). The increase in transporter activity was due to an increased Vmax, and the magnitude of the increase was inversely related to the basal cotransporter activity of the cultures. Increased cotransporter activity was detectable 6 h after exposure, was sustained for 24 h after cells were removed from an albumin-free medium, and was prevented by cycloheximide. Heat-treated BSA, fatty-acid and globulin-free BSA, and gamma-globulins were as effective at increasing Na(+)-glucose cotransporter activity as untreated Fraction V BSA. Dextran, dextran-sulfate, and amino acid supplements were ineffective. Neither protease inhibitors nor chloroquine added to an albumin-containing medium prevented increased alpha-methylglucoside uptake. Albumin did not change the rate of fluid-phase endocytosis in the cultured cells. Na(+)-amino acid cotransport and Na(+)-H+ exchange were either decreased or unchanged after BSA exposure. Exposing apical surfaces of cells grown on permeable membranes to BSA led to a greater increase in activity of the Na(+)-glucose cotransporter relative to controls than did exposing the basolateral surface (145 versus 89%; P < 0.05; N = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

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Fluid phase endocytosis by isolated rabbit lacrimal gland acinar cells

Experimental Eye Research ◽

10.1016/s0014-4835(05)80066-1 ◽

1995 ◽

Vol 60 (5) ◽

pp. 511-525 ◽

Cited By ~ 38

Author(s):

J. Peter Gierow ◽

Robert W. Lambert ◽

Austin K. Mircheff

Keyword(s):

Lacrimal Gland ◽

Acinar Cells ◽

Fluid Phase ◽

Fluid Phase Endocytosis

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