Download PCDU Seminar Myriam Murillo 11 November 2015

Endocytosis
PCDU Seminar
Myriam Murillo
11 November 2015
Content
 Endocytosis functions
 Endomembrane system
 Clathrin-mediated endocytosis
 Clathrin-independent endocytic pathway
Endocytosis Functions
• Engulfing external materials.
• Regulating the abundance and distribution of plasma membrane (PM) proteins and lipids.
• Occurs at the cell surface and involves the formation of membrane invaginations, followed
by vesicle separation from the PM.
• Removal or recycling of materials from the apoplasm or extracellular space.
http://web.stanford.edu/group/virus/1999/jchow/endoem.jpg
Endomembrane system
• Synthesis, sorting, delivery and degradation of macromolecules within the cell.
• Membrane-bound organelles that are connected either directly or through a series of
transport vesicles.
 Endoplasmic reticulum
 Golgi complex and trans-Golgi network (TGN)
 Endosomes
 Lysosomes or vacuoles
Endomembrane system
MVB: Multivesicular Body
CCV: Clathrin-coated Vesicle
NCV: Non-coated Vesicle
TGN: Trans-Golgi Netwrok
Bassham & Contento. (2012). Structure and function of endosomes in plant cells.
Endosomes
• First point of fusion for endocytic vesicles,
used to internalize extracellular materials.
• Transport of materials.
• Provide intermediate compartments where
materials can be stored, sorted and then sent
to a designated target.
• Maintenance of the vacuole, cell growth and
growth of specialized cells such as pollen
tubes.
• Platforms for signaling pathways.
http://zmbe.uni-muenster.de/institutes/imv/schelhaas/schelres.htm
Endocytic pathway in plants
Biosynthetic transport to the
PM through the TGN or MVB.
PM components internalized
into endocytic vesicles to
TGN.
Recycling transport to PM
through a specialized RE.
Degradative transport for
newly synthesized vacuolar
components and cellular
materials destined for
degradation in the vacuole.
Retrograde transport.
Bassham & Contento. (2012). Structure and function of endosomes in plant cells.
Clathrin-mediated endocytosis (CME)
Bednarek, Sebastian Y. (2013). Budding and braking news about clathrin-mediated endocytosis.
Clathrin-coated vesicle (CCV) formation
• Initial step of endocytosis.
• Clathrin: major coat protein made up of a triskelion.
• Triskelia  assembled at the vesicle formation sites on the PM forming a lattice
surrounding the invaginating membrane.
Sigma Aldrich Company. (2015)
CME mechanism
1.
Binding of heterotetrameric adaptor protein 2 (AP-2) complex/clathrin to the PM.
2.
Cargo recognition/recruitment.
3.
Vesicle maturation.
4.
Scission.
5.
Dissociation.
http://www.frontiersin.org/files/Articles/107466/fpls-05-00408-HTML/image_m/fpls-05-00408-g004.jpg
Adaptor protein 2 (AP-2) complex
* Required for auxin
transport,
brassinosteroid
signaling and cellulose
metabolism.
Bednarek, Sebastian Y. (2013). Budding and braking news about clathrin-mediated endocytosis.
Cargo recognition motifs
• Ubiquitination  post-translational modification, which results in the covalent linkage of
an ubiquitin molecule to a lysine residue on a target protein. At PM, cargo molecules can
be ubiquitinated (sorting signal for internalization).
Cargo
molecules
PM
Endosomes
Numerous cellular locations
Proteins destined for
degradation 
internal vesicles of
LEs.
Proteins destined for
recycling  limiting
outer membrane.
Scission by Dynamin and dynamin-related
proteins (DRPs)
• Required for clathrin-mediated trafficking.
• GTPases  tubulate membranes.
• Assembles into helical structures at the neck of clathrin-coated buds and constricts and
pinches off the bud neck membrane.
• DRP1 and DRP2 roles in CCV formation during endocytosis.
http://www.endocytosis.org/Endocytosis/CCVAssembly.html
DRP1A and DRP2B
• Associated with clathrin at the PM.
• Expressed together.
• Cytokinesis  co-localized on the leading edge of the forming cell plate.
• Both begin to accumulate at the vesicle formation sites of the plasma membrane after the
clathrin assembly and detach from there at the same time as or immediately after the
clathrin disassembly.
• Their molecular properties are different.
http://www.cmri.org.au/Research/Research-Units/Cell-Cycle/For-Researchers
Regulation of CME
• Plant developmental processes: polar growth of root hair cells, cell-to-cell communication,
establishment of root cell polarity, and other auxin-dependent processes.
Salicylic
acid (SA)
Strigolactone
Auxin
Clathrin-independent endocytic pathway
• Requires the integrity of sterol-rich membrane microdomains within the PM.
• Flotillin: membrane associated protein. Localized to the PM and endosomal structures.
• Formation of membrane microdomains, membrane invaginations, and vesicle budding.
• Functions in seedling development.
• Arabidopsis  required for optimal growth and forms large endocytic vesicles in root cells.
http://www.nmr.uni-konstanz.de/research.html
Flot1 and Flot2
• Co-assembled  facilitate compartmentalization and functional specialization within the
membrane.
• Interface between signaling receptors  interacting with and modulating the cytoskeleton.
• Controlled by tyrosine residue phosphorylation (kinases).
Benjamin J. Nichols. (2011). The roles of flotillin microdomains-endocytosis and beyond.
Models of flotillin function in endocytosis
No
conclusive
experimental
evidence !
Benjamin J. Nichols. (2011). The roles of flotillin microdomains-endocytosis and beyond.