the endo-lysosomal system

Master program MOLECULAR AND CELLULAR LIFE SCIENCES Block 1: Intracellular membrane processes

11-10-2012: 9:00-10:45 (BBL205) The endo-lysosomal system

Judith Klumperman Department of Cell Biology UMC Utrecht The Netherlands

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Klumperman group :

- Understand how genetic mutations lead to cellular disorganization and disease, with special emphasis on diseases related to the cellular digestive system, the endo-lysosomes

- Development of (electron) microscopy methods: immunoEM, tomography, correlative microscopy

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Department of Cell Biology, UMC Utrecht

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What is endocytosis and why is it important? Entries into the cell Composition of the endo-lysosomal system Transport between endosomal compartments Targeting of newly synthesized lysosomal proteins lysosome-related functions and disorders

The endo-lysosomal system

What do cells do? Cells move

Cells reproduce

Cells eat

Cells communicate

Cells die

Macrophage chasing a bacteria

Cells eat

Source: Alberts, Molecular Biology of the Cell

A bacteria that is eaten by a macrophage ends up in a lysosome

bacteria

Macrophage

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endosomal structures

PM

nucleus

Lysosomes live electron microscopy


Golgi

ER

endosome

endosomes/lysosomes

plasma membrane

ER

intracellular

extracellular environment

mitochondria

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1. essential molecules: nutrients, hormones, growth factors

2. pathogens: viruses, bacteria

3. drugs, medicines

Essential small molecules - amino acids, sugars, ions - traverse the plasma membrane through protein pumps or channels

Uptake of macromolecules occurs via membrane-bound vesicles that form from the plasma membrane => endocytosis

Endocytosis: the main entrance into the cell

Entries into the cell

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Pinocytosis

phagocytosis

process of engulfing solid particles by the cell membrane to form an internal phagosome: e.g. bacteria, dead cells, small particles.

major mechanism to remove pathogens and cell debris

Clathrin-mediated endocytosis

Plasma membrane

cyt cyt cyt cyt

Clathrin coat

Lumen transport vesicle

Endocytosed proteins & lipids

cytosol

Outside the cell

Plas

ma

mem

bran

e

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Formation of clathrin coats requires adaptor proteins

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Adapted from: Braulke and Bonifacino, BBA, 2008

Adaptor complexes involved in clathrin-mediated vesicular transport

TGN-to-endosome endocytosis endosome-to-lysosome

basolateral? APP in neurons?

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The endosomal system

Pathways to the lysosome: endocytosis

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phagophore

autolysosome

autophagosome

Lecture Reggiori

Pathways to the lysosome: Autophagy

Lysosomes can secrete their content

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Lysosomes digest their content to small units that are released into the cytosol and used for de novo syntheses of proteins and lipids

MVB/

MVB = multivesicular body 24

The early endosomal pathway

Early endosome ultrastructure

Bi-layered clathrin coat

Recycling tubule

Inward budding vesicle

ILVs

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Sachse et al., MBC 13, 2002

clathrin10

wt-Rab14-GFP

EE

N

P HepG2: Tf10

Recycling Tf/TfR are found in early endosome associated tubules but do not accumulate in the clathrin coat and do not enter intraluminal vesicles

ILVs

Recycling tubules

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1. Binding Tf and iron (Fe) to TfR

2. Release ferritin in endosome

3. Recycling Tf and TfR

4. Export Fe into cytoplasm

Why is recycling important? Iron uptake via the TfR cycle

P

EE

EE

I: EGFR concentrates in the endosomal clathrin coat II: EGFR concentrates in the

Intraluminal vesicles

HeLa + 5 min epidermal growth factor (EGF): EGFR10

Lysosome-directed epidermal growth factor receptor accumulates in the endosomal clathrin coat before incorporation into intraluminal vesicles

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Why is endosomal sorting important? EGFR signaling and downregulation

Sorting into the intraluminal vesicles of MVBs/late endosomes attenuates EGFR signaling

ESCRT components are identified as tumor suppressors

Adapted from: Tanaka et al., Cancer Science 2008 30

The endosome associated ESCRT complex

Early endosomes: sorting by retention

EGFR is retained in the clathrin coat by the ESCRT complex and incorporated into intraluminal vesicles

TfR is not retained in the coat and follows the constitutive pathway to the recycling endosomes

=> the morphology of the early endosome strongly reflects its function by a sub-division into specialized membrane domains

Sachse et al., MBC, 2004

MVB/

MVB = multivesicular body 32

The late endosomal pathway

MVB/

LE

LYS

Lysosome biogenesis

Maturation of endosomes is reflected by an increasing number of intraluminal vesicles

Figure 2 from Jatta Huotari and Ari Helenius The EMBO Journal online publication doi:10.1038/emboj.2011.286

The endosome/lysosome system

2011 European Molecular Biology Organization. 34

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From: Prior and Luzio, BBA, 2009

Research lecture Casper Jonker


2011 European Molecular Biology Organization.

Late endosome motility.

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Endo-lysosomes are at the crossroad of the endocytic and biosynthetic pathways

LAMP-1

LAMP-2

From: Eskelinen et al., Trends in Cell Biology, 2003

LAMP-3/CD63

LIMP-2

V-type H+-ATPase

~25 lysosomal membrane proteins

Building a functional lysosome

cathepsins

Lysosomal proteins Soluble lysosomal hydrolases => degradation Integral lysosome associated membrane proteins (LAMPs) => structural integrity

Lysosome biogenesis: transport of lysosomal proteins

LAMP-1

LAMP-2

lysosome

cathepsins

Mannose 6-phosphate receptors (MPRs)

=> transport of soluble lysosomal enzymes

Soluble lysosomal hydrolases are transported by mannose 6-phosphate receptors (MPRs)

cathepsins

From: Braulke and Bonifacino, BBA, 2008

MPRs mediate the TGN to endosome transport of the majority of lysosomal (acid) hydrolases

Mannose 6-phosphate receptors (MPRs) are the primary receptors for transport of lysosomal hydrolases

There are more than 50 lysosomal hydrolases which are equipped with a mannose-6-phosphate tag that is recognized by MPRs

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Mannose 6-phosphate receptor (MPR)-dependent transport of soluble lysosomal enzymes

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?

TGN to endosome transport pathways of LAMPs are unknown, but

independent of MPRs

LAMP-1

LAMP-2

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Research in Klumperman group

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General lysosomal functions degradation of extra- & intracellular material => digestion (extra)

=> autophagy (intra)

Lysosomes are the degradative end stations of the endocytic pathway

Melanosomes Pigmentation

Azurophilic granules Defense

Cytolytic granules Defense

MHCIIcompartments Immunity

Weibel-Palade Bloodclotting

Lysosomes are also involved in storage and secretion

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>50 distinct diseases are caused by a defect in lysosome functioning

Worldwide 1:5000 births have a lysosomal disorder

Common symptoms: neuropathologies, kidney malfunctioning, bone deformations, muscle diseases, general failure to thrive

For most lysosome related disorders there is no cure

Lysosome related disorders

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Classes of genetic lysosomal disorders

lysosomal storage/metabolic: >45 pigmentation-bleeding

non-metabolic without a pigmentation defect

Human disorders due to a deficiency in lysosomal function

Secondary lysosomal defects cancer

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LIPIDOSIS SULFATASE MULTIPLE DEFICIT (AUSTIN DISEASE) FABRY * FARBER GAUCHER * KRABBE LANDING LEUCODYSTROPHY METACHROMATIC NIEMANN-PICK * SANDHOFF SCHINDLER TAY-SACHS WOLMAN CHEDIAK-HIGASHI SYNDROM MUCOPOLYSACCHARIDE DISEASES HUNTER * HURLER * MAROTEAUX-LAMY MORQUIO SANFILIPPO SCHEIE SLY MPS IX ABNORMAL LYSOSOMAL TRANSPORT SALLA DISEASE (Sialic acid storage disease) CYSTINOSIS * DANON OLIGOSACCHARIDOSIS DISEASES AND GLYCOPROTEINOSIS DISEASES MUCOLIPIDOSIS TYPE II (I Cell) MUCOLIPIDOSIS TYPE III MUCOLIPIDOSIS TYPE IV ASPARTYLGLUCOSAMINURIA SIALIDOSIS GALACTOSIALIDOSIS FUCOSIDOSIS MANNOSIDOSIS PYCNODYSOSTOSIS GLYCOGEN STORAGE DISORDER SYNDROME TYPE II ( POMPE DISEASE) * CEROD LIPOFUSCINOSIS DISORDERS PAPILLON-LEFEVRES SYNDROME

Lysosomal storage disorders

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Classes of genetic lysosomal disorders

lysosomal storage/metabolic: >45 pigmentation-bleeding

non-metabolic without a pigmentation defect

Human disorders due to a deficiency in lysosomal function

Secondary lysosomal defects cancer

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I-cell disease/ mucolipidosis II (MLII)

GlcNAc-phosphotransferase

Mucolipidosis II (MLII)

or I-cell disease

X

X

GlcNAc phosphotransferase equips lysosomal hydrolases with a M6P-tag that

in the TGN is recognized by MPRs

An increasing number of disorders is attributed to a disease of the lysosomal system..

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Mucolipidosis II (MLII) => growth deficiency, mental retardation,

swollen salivary glands, skeleton distortions

Cellular characteristics: abnormal vacuolization (inclusions) in cells of mesenchymal origin, especially fibroblasts

The most severely affected system is the skeleton

rare disorder: ~1 in 640,000 live births

MLII mouse model (Gelfman et al., 2007. Invest. Ophtalmol. Vis. Sci. 48, 5221-5228)

330531

N m

G

m I-cell B cell: MHCII10

Eline van Meel

Inclusion body

I-Cell B cells contain inclusion bodies, which are a hallmark of I-cell disease

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Children afflicted with mucolipidosis II and III generally are short in stature and have distinctive facial features.

Bone

Osteoclasts are bone resorbing cells that secrete acid hydrolases

Multi nucleated

Resorption lacuna: extracellular lysosome

TGN

Bone

1. Direct secretion 2. Via secretory lysosomes

Possible ways of lysosomal hydrolase secretion by osteoclasts

TGN

Resorption lacuna: extracellular lysosome

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Principles of vesicular transport

Vesicular transport occurs by formation of a transport vesicle from a donor compartment and fusion with target organelle 62

Coats, tethers, SNAREs, Rabs

1. Selection of cargo

2. Formation of a transport vesicle

3. Transport of vesicle towards target compartment

4. Fusion with target compartment

Principles of vesicular transport

establish the initial connection between a vesicle and its target membrane two structurally different groups; multisubunit tethering complexes large coiled-coil tethering proteins.

Tethering proteins

SNAREs

RABs

Stenmark, 2009

Figure 15-21 Essential Cell Biology ( Garland Science 2010)


2011 European Molecular Biology Organization.

The endosome/lysosome system

Lecture_Klumpermann (part1a).pdfSlide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32Slide Number 33Slide Number 34Slide Number 35Slide Number 36Slide Number 37Slide Number 38Slide Number 39Slide Number 40Slide Number 41Slide Number 42Slide Number 43Slide Number 44Slide Number 45Slide Number 46Slide Number 47Slide Number 48Slide Number 49Slide Number 50Slide Number 51Slide Number 52Slide Number 53Slide Number 54Slide Number 55Slide Number 56Slide Number 57Slide Number 58Slide Number 59Slide Number 60Slide Number 61Vesicular transport occurs by formation of a transport vesicle from a donor compartment and fusion with target organelleSlide Number 63Slide Number 64Slide Number 65Slide Number 66Slide Number 67Slide Number 68Slide Number 69

Lecture_Klumpermann (part1b)Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32Slide Number 33Slide Number 34Slide Number 35Slide Number 36Slide Number 37Slide Number 38Slide Number 39Slide Number 40Slide Number 41Slide Number 42Slide Number 43Slide Number 44Slide Number 45Slide Number 46Slide Number 47Slide Number 48Slide Number 49Slide Number 50Slide Number 51Slide Number 52Slide Number 53Slide Number 54Slide Number 55Slide Number 56Slide Number 57Slide Number 58Slide Number 59Slide Number 60Slide Number 61Vesicular transport occurs by formation of a transport vesicle from a donor compartment and fusion with target organelleSlide Number 63Slide Number 64Slide Number 65Slide Number 66Slide Number 67Slide Number 68Slide Number 69

Lecture_Klumpermann (part1c)Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32Slide Number 33Slide Number 34Slide Number 35Slide Number 36Slide Number 37Slide Number 38Slide Number 39Slide Number 40Slide Number 41Slide Number 42Slide Number 43Slide Number 44Slide Number 45Slide Number 46Slide Number 47Slide Number 48Slide Number 49Slide Number 50Slide Number 51Slide Number 52Slide Number 53Slide Number 54Slide Number 55Slide Number 56Slide Number 57Slide Number 58Slide Number 59Slide Number 60Slide Number 61Vesicular transport occurs by formation of a transport vesicle from a donor compartment and fusion with target organelleSlide Number 63Slide Number 64Slide Number 65Slide Number 66Slide Number 67Slide Number 68Slide Number 69

Lecture_Klumpermann (part1d)Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32Slide Number 33Slide Number 34Slide Number 35Slide Number 36Slide Number 37Slide Number 38Slide Number 39Slide Number 40Slide Number 41Slide Number 42Slide Number 43Slide Number 44Slide Number 45Slide Number 46Slide Number 47Slide Number 48Slide Number 49Slide Number 50Slide Number 51Slide Number 52Slide Number 53Slide Number 54Slide Number 55Slide Number 56Slide Number 57Slide Number 58Slide Number 59Slide Number 60Slide Number 61Vesicular transport occurs by formation of a transport vesicle from a donor compartment and fusion with target organelleSlide Number 63Slide Number 64Slide Number 65Slide Number 66Slide Number 67Slide Number 68Slide Number 69

Lecture_Klumpermann (part2)Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32Slide Number 33Slide Number 34Slide Number 35Slide Number 36Slide Number 37Slide Number 38Slide Number 39Slide Number 40Slide Number 41Slide Number 42Slide Number 43Slide Number 44Slide Number 45Slide Number 46Slide Number 47Slide Number 48Slide Number 49Slide Number 50Slide Number 51Slide Number 52Slide Number 53Slide Number 54Slide Number 55Slide Number 56Slide Number 57Slide Number 58Slide Number 59Slide Number 60Slide Number 61Vesicular transport occurs by formation of a transport vesicle from a donor compartment and fusion with target organelleSlide Number 63Slide Number 64Slide Number 65Slide Number 66Slide Number 67Slide Number 68Slide Number 69

Lecture_Klumpermann (part3a)Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32Slide Number 33Slide Number 34Slide Number 35Slide Number 36Slide Number 37Slide Number 38Slide Number 39Slide Number 40Slide Number 41Slide Number 42Slide Number 43Slide Number 44Slide Number 45Slide Number 46Slide Number 47Slide Number 48Slide Number 49Slide Number 50Slide Number 51Slide Number 52Slide Number 53Slide Number 54Slide Number 55Slide Number 56Slide Number 57Slide Number 58Slide Number 59Slide Number 60Slide Number 61Vesicular transport occurs by formation of a transport vesicle from a donor compartment and fusion with target organelleSlide Number 63Slide Number 64Slide Number 65Slide Number 66Slide Number 67Slide Number 68Slide Number 69

Lecture_Klumpermann (part3b)Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32Slide Number 33Slide Number 34Slide Number 35Slide Number 36Slide Number 37Slide Number 38Slide Number 39Slide Number 40Slide Number 41Slide Number 42Slide Number 43Slide Number 44Slide Number 45Slide Number 46Slide Number 47Slide Number 48Slide Number 49Slide Number 50Slide Number 51Slide Number 52Slide Number 53Slide Number 54Slide Number 55Slide Number 56Slide Number 57Slide Number 58Slide Number 59Slide Number 60Slide Number 61Vesicular transport occurs by formation of a transport vesicle from a donor compartment and fusion with target organelleSlide Number 63Slide Number 64Slide Number 65Slide Number 66Slide Number 67Slide Number 68Slide Number 69

Lecture_Klumpermann (part3c)Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32Slide Number 33Slide Number 34Slide Number 35Slide Number 36Slide Number 37Slide Number 38Slide Number 39Slide Number 40Slide Number 41Slide Number 42Slide Number 43Slide Number 44Slide Number 45Slide Number 46Slide Number 47Slide Number 48Slide Number 49Slide Number 50Slide Number 51Slide Number 52Slide Number 53Slide Number 54Slide Number 55Slide Number 56Slide Number 57Slide Number 58Slide Number 59Slide Number 60Slide Number 61Vesicular transport occurs by formation of a transport vesicle from a donor compartment and fusion with target organelleSlide Number 63Slide Number 64Slide Number 65Slide Number 66Slide Number 67Slide Number 68Slide Number 69



the endo-lysosomal system

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