Lecture #10

Question 1

what are lysosomes?

Answer 1

ubiquitous organelles located in the cytoplasm

Question 2

what are the functions of lysosomes?

Answer 2

involved in autophagy, secretion, in the repair of the pm and in the energy control metabolism in the cell

Question 3

what are some examples of diseases associated to lysosomal dysfunction?

Answer 3

Alzheimers, Parkinsons, MS

Question 4

what is an example of a cell type that is more sensitive to lysosomal dysfunction?

Answer 4

neurons

Question 5

what type of organelles are lysosomes?

Answer 5

intracellular organelles with a very acidic lumen - lysosomal enzymes work in an acidic pH

Question 6

where are the lysosomes located?

Answer 6

in the perinuclear space - there are different patterns depending on the cell stage (can either be perinuclear or very far from the nucleus)

Question 7

describe the shape of lysosomes:

Answer 7

either spherical or tubular → size and number are quite heterogeneous depending on the stage of the cell cycle and the type of cell

Question 8

describe lysosome biogenesis:

Answer 8

lysosomal enzymes follow the normal protein synthesis in the cell, then they are packaged into vesicles in the golgi, are modified, and delivered to the lysosomes via a specific tag

Question 9

what is lysosomal biogenesis regulated by?

Answer 9

many intracellular and extracellular signals → the state of the cell impacts biogenesis

Question 10

describe the membrane of the lysosome:

Answer 10

a phospholipid bilayer membrane rich in carbohydrates

Question 11

describe the lumen of the lysosome:

Answer 11

there are many soluble hydrolyses as well as activators

Question 12

describe the other membrane formations in the lysosome:

Answer 12

interfaces between the intra-lysosomal vesicles are important because some enzymes are bound to these intramembrane formations

Question 13

describe the lysosomal proteins:

Answer 13

these are not only hydrolyses and activators, but also intramembrane or membrane-associated proteins such as LYNUS

all proteins are classified according to their function (structural proteins, channels, protein that regulate fusion, catabolic enzymes, transporters, etc)

Question 14

what is the primary function of the lysosomes?

Answer 14

the degradation of substrates

Question 15

where does the primary function of lysosomes occur?

Answer 15

on the surface of the intra-lysosomal vesicles catalyzed by hydrolyses

Question 16

about how many hydrolyses are located in the lumen?

Answer 16

around 70

Question 17

describe the cycle of lysosomes:

Answer 17

1. entry of the material into the lysosomes either from the inside or the outside of the cell
2. degradation of the material resulting in the formation of building blocks
3. building blocks are recycled

*must be tightly regulated in order to maintain the correct energy metabolism in the cell

Question 18

describe the organization of lysosomes:

Answer 18

they are part of a membrane system (sometimes it is difficult to see the boundaries between different membrane compartments such as the golgi, vesicles, endosomes, etc)

Question 19

how are hydrolyses driven to the lysosomes?

Answer 19

they have specific tags → in the golgi they are tagged with specific molecules in particular mannose 6 phosphate (M6P) which is recognized by M6P receptors

Question 20

do all enzymes get delivered directly to the lysosomes?

Answer 20

no - a fraction of the enzymes escape and are secreted directly outside the cell

Question 21

what happens to the enzymes that escape?

Answer 21

since they are tagged with M6P they can be re-uptaken by neighboring cells that express M6P receptors → cross correction

Question 22

what is enzyme replacement therapy?

Answer 22

based off of the cross correction system: if there is an enzyme deficiency, the functional copy can be provided by healthy cells or by gene therapy → the secreted enzymes can be recaptured by the deficient cells that do not produce the enzyme themselves

Question 23

describe the endocytic route:

Answer 23

plasma membrane - early endosomes - lysosome - recycled back to plasma membrane

Question 24

what is the pH of the lysosome?

Answer 24

5

Question 25

what is an example of a lysosomal product where transporters are necessary?

Answer 25

cholesterol

Question 26

how is cholesterol formed?

Answer 26

the lysosome degrades lipoprotein complexes (proteins and lipids) releasing cholesterol to be transported out of the organelle into the cytosol

Question 27

in what types of cells do lysosomes exert exocytosis function in order to get rid of toxic material or aggregates?

Answer 27

secreting cells such as hematopoietic cells, osteoclasts, melanocytes, or in non-physiological conditions

Question 28

how does the lysosome function when the cells are in a state of starvation?

Answer 28

they can sense the lack of nutrients and activate a response that provides the cells with nutrients through lysosomal-mediated degradation and autophagy

Question 29

how does the lysosome function when the cells are in an anabolic state?

Answer 29

they can synthesize proteins and lipids to shut down autophagy

Question 30

how many different forms of hydrolyses does the lysosome contain?

Answer 30

70

Question 31

how is the pH of the lysosome maintained?

Answer 31

there is a hydrogen pump that carries out active transport, and imports H+ ins inside the lysosome against the concentration gradient

Question 32

describe the proteins that are embedded in the membrane of the lysosomes:

Answer 32

complexed with sugars which face the organelle lumen to protect the membrane from the acidic environment

Question 33

what is the function of LYNUS?

Answer 33

huge complex of proteins that serves as the lysosomal nutrient sensing machinery

Question 34

what are the two types of channels in lysosomes?

Answer 34

CIC7 (chloride) and MCOLN1 (muco-lipin)

Question 35

describe the function of CIC7:

Answer 35

helps the hydrogen pumps make the environment more acidic

Question 36

describe the function of MCOLN1?

Answer 36

a calcium channel which regulates the efflux of the calcium

Question 37

what other channels are important for calcium import?

Answer 37

TPC1-2 : two-pore channels that assist the entry and exit of calcium

Question 38

what is the category of trafficking and fusion machinery?

Answer 38

SNARES

Question 39

in order for a vesicle to fuse with the lysosome, what must be true?

Answer 39

their membranes must be compatible in terms of composition and membrane identity

Question 40

what is the player of recognition between vesicles, endosomes, or autophagosomes which fuse with the lysosome?

Answer 40

RAB7

Question 41

what is the name of the group of signaling lipids necessary, and what specifically is the key master regulator of fusion with the lysosome?

Answer 41

phosphoinositides ; phosphatidylinositol-3,5-bisphosphate

Question 42

which are the three SNARE proteins that allows for fusion?

Answer 42

vSNARE, tSNARE, and VAMP

Question 43

what type of enzymes are found on the surface and in the lumen that are devoted to degradation and catabolism?

Answer 43

catabolic enzymes

Question 44

what is the function of LAMP2A?

Answer 44

transporter that mediates chaperone-mediated autophagy

Question 45

what are the two different routes of autophagy?

Answer 45

membrane (macroautophagy) and chaperone-mediated

Question 46

how do proteins brought by chaperone-mediated autophagy interact with the lysosome?

Answer 46

pass from the cytosol, and are incorporated into the lysosome directly without fusion

Question 47

what must proteins contain in order to go through chaperone-mediated autophagy?

Answer 47

KFREQ specific pentapeptide

Question 48

what chaperone recognizes proteins that need to be degraded by this pathway, and brings them to the lysosome?

Answer 48

HSPA8

Question 49

what specific transporter is involved in cholesterol export into the cytosol?

Answer 49

NCP1

Question 50

how can LAMP1 be used in the laboratory?

Answer 50

it is a protein that can be used to recognize lysosomes became it is an integral structural protein embedded into the membrane

Question 51

what is the fusion of the lysosomes and the pm dependent on?

Answer 51

calcium - dependent

Question 52

what channels (among others) release calcium in relation to lysosomal-pm binding?

Answer 52

MCLON1 and TRPML1

Question 53

in regards to the fusion of the lysosome and the pm, which snare proteins are present on the lysosome that mediate this interaction?

Answer 53

V-SNARE and VAMP7

Question 54

in regards to the fusion of the lysosome and the pm, which snare proteins are present on the pm that mediate this interaction?

Answer 54

T-SNARE, SNAP23, and syntaxin4

Question 55

in what type of cells is it common for membrane repair to be needed?

Answer 55

muscle cells - constantly exposed to mechanical stress

Question 56

what is the interaction between the lysosome and membrane repair regulated by?

Answer 56

calcium and the protein Dysferlin → must have both

Question 57

what are dysferinopathies?

Answer 57

muscular dystrophies or other atrophies of muscle cells due to the apoptosis as a result of continuous damage (often pediatric onset)

Question 58

what are calpains?

Answer 58

proteases that digest the actin cortical cytoskeleton temporarily to allow for vesicle fusion

Question 59

what stage of endoscope interacts with the lysosome?

Answer 59

late endosome → forms the endolysosome

Question 60

what occurs after digestion in the endolysosome?

Answer 60

the lysosome buds off again from the endolysosome to form a new lysosome

Question 61

what occurs after the autophagosome and the lysosome fuse?

Answer 61

they become the autolysosome and degrade the material due to the acidic pH, then there is lysosomal reformation

Question 62

what process is extracellular material digested by?

Answer 62

formation of multi-vesicular bodies forming the late endosome and then finally joining the lysosome to become the endolysosome

Question 63

what is chaperone-mediated autophagy recognized by on the lysosome?

Answer 63

LAMP2A

Question 64

where do lysosomal hydrolyses come from?

Answer 64

1. synthesized in the ER 2. phosphorylated in the cis gogli to attach a mannose and phosphate forming the m6p tag 3. go through several cisterns to reach the trans golgi 4. bud off in clathrin-coated vesicles

Question 65

what two reasons is Claritin coating fundamental?

Answer 65

to interact with actin (light chain) and Bar proteins that help the pm curve, and to select the cargo that needs to be transported

Question 66

what does clathrin work with in order to be able to perform its function?

Answer 66

adaptors like AP2 which recognize receptors that are embedded in the vesicle

Question 67

what happens after the clathrin-coated vesicle is delivered?

Answer 67

clathrin / COPI / COPII are recycled back to the ER

Question 68

describe the concept of cross-correction:

Answer 68

if you put two cells close to each other, the WT can cross correct the deficient cell through the exocytosis of the enzyme which is recognized by the receptor of the deficient cell

Question 69

when treating lysosomal deficiency disorders, what cells are used for engineering,?

Answer 69

hematopoietic cells → precursors of monocytes. macrophages which are able to colonize every tissue of the body

Question 70

why are hematopoietic cells used as the vessel?

Answer 70

in a condition where there are not as many lysosomal enzymes present, there is a lot of inflammation so the environment favors the recruitment of monocytes and macrophages

Question 71

what are caveolae?

Answer 71

patches in the pm of high density proteins and lipids. that different from clathrin, are not formed dynamically when needed but are always present

Question 72

what are some examples of selective autophagy?

Answer 72

mitophagy, xerophagy, pexophagy, aggrephagy

Question 73

where are the two main areas where membranes are sourced for autophagy?

Answer 73

the pm or ER

Question 74

name some of the positive regulators of autophagy:

Answer 74

Beclin 1, Vps34 (a subunit that regulates PA trikinase class 3), UVRAG without these proteins the autophagosome cannot be formed and engulfment doesn't occur

Question 75

which ATG proteins are important for autophagosome elongation, and are needed to select cargo, mature, and go through the lysosome?

Answer 75

ATG 5, 7, 12

Question 76

what is the anterograde movement mediated by?

Answer 76

kinesics that move by hydrolyzing ATP

Question 77

what is used for transport from the periphery to the perinuclear region?

Answer 77

dyneins

Question 78

what is basal autophagy and what is an example?

Answer 78

when the ER is too big, so part of it needs to be degraded

Question 79

how are the different autophagic states regulated?

Answer 79

SLCA1 pathway