Nullin Flashcards
1
Q
Why is bringing proteins to the membrane important for cellular function?
A
Bringing proteins to the membrane drives downstream signaling, which is essential for regulating cellular processes like proliferation and differentiation.
2
Q
How do receptors control the localization of proteins?
A
Receptors, when activated by ligands, interact with proteins and drive their localization to the membrane. This is often achieved through binding to phosphorylated residues or through lipid tethers.
3
Q
How does RAS control its localization to the membrane?
A
RAS is lipid-anchored to the plasma membrane via a lipid tether, and its localization to the membrane is a key mechanism controlling its signaling function, particularly in proliferation.
4
Q
What is the role of PtdIns in membrane localization?
A
PtdIns signaling changes the lipid composition of the membrane, creating specific binding sites for proteins with lipid-binding domains, thus driving their localization to the membrane after activation by receptors.
5
Q
How does protein localization affect downstream signaling?
A
The location of a protein in the cell affects which signaling pathways it activates. For example, RAS can activate different pathways depending on its membrane compartment, such as the ERK pathway in the plasma membrane and the JNK kinase stress response in the endoplasmic reticulum.
6
Q
What happens when RAS is moved to different membrane compartments?
A
Moving RAS to different compartments, like the Golgi, activates different downstream pathways, such as the ERK pathway or PI3K pathway, altering cellular responses to signals.
7
Q
What role does the PI3K pathway play in cancer?
A
The PI3K pathway is critical for cell growth and survival. Mutations in PI3K or PTEN can lead to constitutive activation of the pathway, driving uncontrolled cell growth and contributing to cancer.
8
Q
How does PI3K activate downstream signaling?
A
PI3K phosphorylates PIP2 to create PIP3, which then binds to signaling proteins like PKB kinase. This activates a cascade of phosphorylation events, promoting cell survival and growth
9
Q
How does the PTEN phosphatase regulate the PI3K pathway?
A
PTEN dephosphorylates PIP3 back to PIP2, serving as a negative regulator of the PI3K pathway. Mutations in PTEN can lead to uncontrolled signaling through PI3K.
10
Q
How do mutations in PI3K contribute to cancer?
A
Specific mutations in PI3K lead to constitutive activation of the pathway, resulting in excessive production of PIP3, which drives PKB kinase activity and tumorigenesis.
11
Q
How do mutations in PTEN contribute to cancer?
A
PTEN mutations lead to loss of its phosphatase function, preventing the conversion of PIP3 back to PIP2, which results in continued activation of the PI3K pathway and promotion of cancer cell survival.
12
Q
How does PKB kinase mutation affect cancer progression?
A
A mutation in PKB kinase (e.g., E17 glutamate to lysine) causes a charge reversal, enabling PKB to bind PIP2 instead of PIP3, leading to oncogenic signaling and promoting cancer growth.
13
Q
How does the insulin receptor system regulate signaling?
A
Insulin binding to its receptor activates PI3K, which generates PIP3. PIP3 activates PKB kinase, initiating a signaling cascade that regulates various cellular processes like glucose uptake.
14
Q
How do tumor cells alter insulin receptor signaling?
A
Tumor cells often acquire mutations that constitutively activate PI3K, leading to persistent production of PIP3 and continuous activation of PKB kinase, contributing to uncontrolled cell growth and cancer.
15
Q
What does PIP signaling provide for cells?
A
PIP signaling transduces environmental changes into changes in cell behavior, creates novel membrane surfaces for protein interactions, and modulates nuclear surfaces to transduce environmental signals into epigenetic changes.
16
Q
How do phosphoinositides influence membrane surfaces?
A
Phosphoinositides provide highly regulated novel surfaces that can flag and identify different membrane types, and they also modulate protein interactions and regulation at the membrane.
17
Q
What is the role of phosphoinositides in cell signaling?
A
Phosphoinositides act as receptor- and environment-regulated messengers that modulate location and interaction of proteins on membranes, facilitating changes in cellular functions in response to signals.
18
Q
How are phosphoinositides structurally related to lipid molecules?
A
Phosphoinositides are lipids with two fatty acid chains (18-24 carbon atoms) and double bonds, which allow them to play a role in membrane dynamics and protein binding.
19
Q
What is the basic structure of phosphoinositides?
A
Phosphoinositides are composed of a glycerol backbone, two fatty acid chains, and an inositol head group that is linked to a phosphate group. The phosphate group is crucial for their role in signaling.
20
Q
What is the significance of the inositol head group in phosphoinositides?
A
The inositol head group is attached to a glycerol molecule via a diester bond, and it plays a key role in phosphorylation/dephosphorylation processes that regulate membrane interactions and signaling pathways.
21
Q
How do phosphoinositides interact with the membrane?
A
The diester glycerol sits in the membrane, while the inositol head group projects outside, where it is subject to phosphorylation or dephosphorylation, altering its function in membrane dynamics and protein recruitment.
22
Q
How does phosphorylation/dephosphorylation of phosphoinositides regulate cellular processes?
A
The phosphorylation and dephosphorylation of phosphoinositides on the inositol head group create different membrane surfaces that regulate protein recruitment, signal transduction, and location modulation of proteins at the membrane.
23
Q
How does phosphoinositide signaling affect epigenetic regulation?
A
Phosphoinositides can create novel nuclear surfaces that directly transduce environmental signals to the nucleus, influencing epigenetic processes like gene expression and chromatin remodeling.
24
Q
What is the role of phosphoinositides in response to environmental changes?
A
Phosphoinositides act as environment-regulated messengers, enabling cells to adapt to changing environments by altering membrane properties, protein localization, and nuclear signaling pathways.
25
What is the parental lipid in phosphoinositides?
The parental lipid in phosphoinositides is phosphatidylinositol, which consists of a glycerol backbone, two fatty acid chains, and an inositol head group without a phosphate group. The phosphate group is added in various positions to create different phosphoinositides.
26
How does the position of phosphate groups affect phosphoinositide signaling?
The addition of phosphate groups at different positions (such as 3, 4, or 5) on the inositol ring generates distinct phosphoinositides that act as signaling molecules with specific roles in membrane dynamics and protein interaction.
27
Which positions on phosphoinositides can be phosphorylated?
Phosphoinositides can be phosphorylated at the 3, 4, and 5 positions on the inositol ring.
28
What are examples of bis-phosphorylated phosphoinositides?
Examples of bis-phosphorylated phosphoinositides include PI(3,4)P2, PI(4,5)P2, and PI(3,5)P2.
29
What happens when all three positions on phosphoinositides are phosphorylated?
When all three positions are phosphorylated, the result is PI(3,4,5)P3, a key lipid in intracellular signaling.
30
Why isn't the 6th position of phosphoinositides phosphorylated?
The 6th position of phosphoinositides isn't phosphorylated because it is adjacent to the membrane, and its negative charge would cause repulsion due to the membrane's negative charge.
31
What is the role of PIP2 in signal transduction?
PIP2 acts in signal transduction by being cleaved by phospholipase C upon receptor activation, generating two second messengers: DiPIP3 and diacylglycerol (DAG). These regulate calcium signaling and PKB (protein kinase B) signaling.
32
How do lipids act as intracellular messengers?
Lipids like PIP2 act as intracellular messengers by being phosphorylated and interacting with proteins, leading to changes in signaling pathways.
33
Who first discovered the activation of lipids by receptor signaling?
Mabel and Lowell Hokin discovered lipid activation in 1953, showing that acetylcholine activated lipids in the pancreas, leading to their phosphorylation.
34
What happens when phosphoinositides are hydrolyzed by phospholipase C?
Phospholipase C cleaves PIP2 at the phosphodiester bond, generating diacylglycerol (DAG) and IP3. DAG stays in the membrane, while IP3 enters the cytoplasm to activate calcium signaling.
35
How does IP3 activate calcium signaling?
IP3 binds to the IP3 receptor on the endoplasmic reticulum, causing calcium release into the cytoplasm.
36
What role does DAG play in signaling?
DAG binds to the C1 and C2 domains of protein kinase C (PKC), translocating PKC to the membrane and driving downstream signaling.
37
How do viral oncogenes transform cells using PIP2?
Viral oncogenes, like the middle T antigen, activate lipid kinases, resulting in PIP2 phosphorylation and PIP3 generation, which transforms the cell and drives cancer development.
38
What was discovered about PIP3 in the context of cancer research?
It was discovered that PIP3 is not a substrate for phospholipase C but is generated by PIP3 kinase and plays a key role in cellular transformation during cancer development.
39
What experimental approach helped identify proteins that bind to phosphoinositides?
The fishing experiment involved using phosphoinositide-bound beads to capture and identify proteins that interact with phosphoinositides, revealing that 400 proteins bind to PtdIns species in cells.
40
How do phosphoinositides regulate signaling pathways?
Phosphoinositides create membrane surfaces that modulate protein interactions with the membrane, leading to signaling events that regulate cellular functions.
41
How are genetically encoded fluorescent proteins used to study phosphoinositides?
GFP (green fluorescent protein) can be fused to a phosphoinositide-binding domain, allowing researchers to track the localization of specific phosphoinositides like PIP2, PIP3, and PIP4 in live cells.
42
Where are phosphoinositides located in cells?
PIP2 is enriched at the plasma membrane.
PIP4 is found in the Golgi apparatus.
PIP3 is enriched in early endosomes.
43
How does colocalization of phosphoinositides with proteins help?
It helps understand protein complexes, such as in early endosomal fusion or identifying proteins that do not localize in particular compartments.
44
What is PIP3 and how does it function?
PIP3 is a second messenger that does not act as a substrate for phospholipase C. It is important in signaling pathways that involve cellular responses to extracellular signals.
45
What is the role of phosphoinositides in endosomal systems?
PIP3 is enriched in the endosomal system, which plays a role in vesicular trafficking. The levels of enzymes for making and breaking phosphoinositides change as they traffic through the system.
46
How are PIP lipids interconverted in cells?
PIP lipids are interconverted through the action of kinases (which add phosphates) and phosphatases (which remove phosphates). This interconversion can lead to chain reactions of lipid alterations, influencing various cellular processes.
47
What impact do mutations in lipid kinases and phosphatases have?
Mutations in lipid kinases and phosphatases have been found in human diseases, but the exact mechanisms and effects are still not fully understood.
48
How many lipid and inositol messengers can be generated?
The system can generate 7 lipid messengers and 63 other inositol messengers through the interconversion of phosphoinositides.
49
What role do lipid-interacting domains play in phosphoinositide signaling?
Lipid-interacting domains transduce phosphoinositide signals into functional outputs by interacting with phosphoinositides and altering downstream cellular pathways.
50
How do phosphoinositides help define membrane identity in cells?
Phosphoinositides enrich certain lipids in different regions of the cell, helping to define membrane identity and regulate function based on intracellular and extracellular cues.
51
How do cells use phosphoinositides to regulate signaling?
Cells generate distinct pools of phosphoinositides that interact with proteins, resulting in functionally diverse outputs and regulating different pathways in response to specific signals.
52
What is the function of PIP5K?
PIP5K (phosphoinositide-5-kinase) converts PI4P to PtdIns(4,5)P2, a crucial lipid involved in cell signaling.
53
How is PIP5K related to cancer?
In many tumors, PIP5K is upregulated but not mutated. High PIP5K levels correlate with poor prognosis and shorter disease-free survival in cancer patients.
54
Why is PIP5K considered a potential pharmaceutical target?
PIP5K is important for regulating various cellular processes, and inhibiting it could suppress tumor growth. However, inhibiting PIP5K could also cause significant cellular dysfunction, making it a challenging target for drug development.
55
How many isoforms of PIP5K exist, and what are their functions?
There are 3 isoforms of PIP5K:
Alpha is located on the membrane and in the nucleus, possibly involved in epigenetic signaling.
Beta is found on endomembranes.
Gamma is localized to focal adhesions, where it regulates cell-extracellular matrix interactions, actin polymerization, cell polarization, and survival signaling.
56
How does PIP5K regulate signaling in focal adhesions?
In focal adhesions, PIP5K gamma interacts with integrins to control actin dynamics, influencing cell movement, polarization, and survival signaling.
57
What is the relationship between PIP5K and protein kinases?
PIP5K functions similarly to protein kinases, with specificity determined by its loops, which dictate interactions with specific phosphoinositides and downstream signaling pathways.
58
How does actin polymerization drive cell migration?
Actin polymerization pushes against the plasma membrane, forming protrusions at the leading edge of the cell, which helps drive cell movement forward.
59
What role does actin play in morphogenetic processes like neuronal outgrowth?
In processes like neuronal outgrowth, actin polymerization contributes to membrane protrusions and cell elongation, without the need for retraction of the trailing edge.
60
How does actin polymerization contribute to endocytosis?
During endocytosis, actin polymerization generates membrane invaginations and provides the force needed for scission of the endocytic vesicles from the plasma membrane.
61
What is the structure of actin filaments?
Actin filaments consist of polar, helical structures formed from actin monomers (G-actin), with two distinct ends:
Barbed end: The fast-growing end.
Pointed end: The slow-growing end.
62
What is actin filament treadmilling?
Treadmilling refers to the dynamic process where actin subunits are added at the barbed end of the filament and dissociate at the pointed end, driven by ATP hydrolysis, and it plays a central role in actin-based motility.
63
How do phosphoinositides like PIP2 regulate actin dynamics?
PIP2 is involved in regulating actin polymerization, but the exact mechanisms are still not fully understood. PIP2 can influence various actin-related proteins, including:
Profilin and gelsolin, which interact with actin filament ends.
Capping proteins that control the addition and dissociation of actin monomers.
64
What are the three general models for the regulation of actin dynamics?
Exposure of barbed ends after dissociation of capping proteins, such as gelsolin and profilin, allowing for polymerization.
Nucleation of new filaments from monomers by proteins like the Arp2/3 complex.
Fragmentation-induced barbed end generation by proteins such as Cofilin, which severs actin filaments.
65
How does PIP2 regulate actin dynamics in these models?
PIP2 regulates the uncapping of actin filament ends, enabling the polymerization of actin and the nucleation of new filaments, by interacting with actin-binding proteins like profilin and gelsolin.
66
How does PIP2 impact actin polymerization?
PIP2 accelerates actin polymerization by releasing profilin, a protein that shuttles monomeric actin to the growing actin filaments. This allows profilin to pick up more actin monomers, speeding up the polymerization process.
67
How does profilin influence actin polymerization?
Profilin shuttles monomeric actin to the filament ends for polymerization but also inhibits polymerization at high concentrations. PIP2 can overcome this inhibition, allowing profilin to continue its role in actin polymerization.
68
What experimental approach was used to study the effect of PIP2 on actin polymerization?
In a simple experiment, fluorescently labeled actin was used to track changes in fluorescence as actin polymerized. This allowed the researchers to observe how PIP2 enhanced the rate of polymerization.
69
What are focal adhesions?
Focal adhesions are specialized protein complexes that form at the cell membrane where actin filaments interact with the extracellular matrix. They help anchor the cell and mediate signaling for processes like migration and adhesion.
70
How does actin polymerization influence focal adhesion formation?
Actin polymerization provides the mechanical force required for focal adhesion formation, which is essential for cell migration, polarization, and interaction with the extracellular matrix.
71
What are the key interactions between profilin and PIP2?
Profilin interacts with PI(4,5)P2 through two distinct regions. One region overlaps with the actin-binding surface, while the other overlaps with the poly-L-proline binding site. This allows profilin to shuttle between the membrane-bound and actin-bound forms in response to changes in PIP2 concentration, thereby acting as a mediator of external signals to microfilaments.
72
How do phosphoinositides like PIP2 regulate gelsolin activity?
PIP2 inhibits the interaction between gelsolin and F-actin and promotes the dissociation of gelsolin from the barbed end of actin filaments. This regulation is essential for actin remodeling and cell motility.
73
What is the role of actinin in cell motility?
Actinin is an actin-binding protein that forms antiparallel dimers, capable of bundling and crosslinking actin filaments. In response to PIP2, actinin's bundling activity is inhibited, thus playing a role in regulating actin dynamics.
74
What is the role of the Arp2/3 complex in actin polymerization?
The Arp2/3 complex is a nucleator for actin filament formation, consisting of 7 subunits, including ARP2 and ARP3, which resemble monomeric actin. The complex binds to existing actin filaments and promotes the formation of branched actin structures.
75
How is the actin nucleating activity of the Arp2/3 complex regulated?
The WASP family proteins (WASP, N-WASP, WAVE, WASH) stimulate Arp2/3 complex activity. The V-domain of WASP proteins interacts with actin monomers, while the VCA domain associates with Arp2/3, leading to nucleation and branching of actin filaments.
76
What biological processes depend on the Arp2/3 complex?
The Arp2/3 complex is required for various processes such as cell migration, phagocytosis, endosome-to-lysosome trafficking, and mitochondrial movement. It also plays a role in pathogen-induced actin movement, as seen during infection by Listeria and Shigella.
77
How is cell migration involved in cancer?
Cell migration is a key process in tumor metastasis, where cancer cells move from the primary tumor site to other parts of the body. This movement is driven by the formation and breakdown of focal adhesions (FAs) that allow cells to interact with the extracellular matrix (ECM) and migrate.
78
What is the role of focal adhesions (FAs) in cell migration?
Focal adhesions are protein complexes that link integrins on the plasma membrane to the actin cytoskeleton. These complexes play a crucial role in cell adhesion, migration, and signal transduction. The formation and turnover of FAs enable cells to move by attaching to the ECM at the front and releasing at the rear.
79
How does the lamellipodium contribute to cell migration?
The lamellipodium is a thin, sheet-like extension at the leading edge of the cell formed by actin polymerization. It provides the force for membrane protrusion, and as focal adhesions form at the front and are broken down at the back, the cell moves forward.
80
How do integrins contribute to focal adhesion formation?
Integrins are transmembrane receptors that bind to the extracellular matrix (ECM) and interact with intracellular proteins such as talin. This interaction drives focal adhesion formation, which in turn impacts actin dynamics, cell motility, and signal transduction.
81
What role does talin play in focal adhesion formation?
Talin is essential for focal adhesion (FA) formation, acting as a recruitment platform for intracellular proteins. It plays a critical role in mediating actin dynamics, proliferation, and cell survival through the recruitment of signaling components.
82
How do integrins regulate inside-out signaling?
The inside-out signaling mechanism involves the interaction of integrins with intracellular proteins like talin, which helps activate integrins and drives the formation of focal adhesions. This process is crucial for cell adhesion and migration.
83
What is the structural composition of talin?
Talin is a large 270 kDa protein with 18 domains. It has a ~50 kDa globular head, a long rod composed of 62 helices forming 13 helical bundle domains (R1-R13).
84
How does talin exhibit spring-like behavior?
Talin undergoes a unique conformational change, unfolding into a linearly elongated 60-100 nm rod-like shape. This allows it to bind to multiple focal adhesion components, including vinculin and actin.
85
What is the role of the FERM domain in talin?
The FERM domain (4.1-ezrin-radixin-moesin domain) is located in the head of talin and contains 4 subdomains (F0-F3). It is responsible for integrin binding and interaction with PI(4,5)P2, enabling talin's regulated attachment to the membrane.
86
How do specific residues in talin contribute to its interaction with PIP2?
Residues in the FERM domain—including K272, K316, K324, E342, and K343—are responsible for talin's interaction with PI(4,5)P2, which regulates its activation and membrane attachment.
87
How does RIAM regulate talin activation?
RIAM is an effector of RAP1, binding to talin and driving a conformational switch that activates talin from its autoinhibited state. This process is essential for the activation of integrins and focal adhesion formation.
88
What is the role of PIP2 in talin activation?
PIP2 interacts with the FERM domain of talin, inhibiting the head-tail interaction of talin, which is required for talin activation. The interaction with RIAM and RAP1 also aids in talin activation, allowing it to bind to the membrane and activate integrins.
89
How does the combination of RAP1, RIAM, and PIP2 activate talin?
The binding of RAP1 to RIAM activates the conformational switch in talin, which is further facilitated by PIP2 interaction. This leads to the unfolding of talin and its membrane association, enabling integrin activation and the formation of focal adhesions.
90
How does PIP2 influence integrin activation and focal adhesion formation?
PIP2 binds to talin's FERM domain, inducing a conformational change that activates talin. This activation allows talin to interact with integrins, driving inside-out signaling and the formation of focal adhesions.
91
What is the role of PIP2 in oligomerization and activation of vinculin?
PIP2 promotes the oligomerization of vinculin, which is essential for vinculin's role in focal adhesion dynamics. The interaction between vinculin and PIP2 is critical for actin bundling and focal adhesion stability.
92
What happens when vinculin mutations disrupt PIP2 binding?
Mutations in vinculin that disrupt the interaction with PIP2 (e.g., changes in R945, K944, and 10161) completely attenuate vinculin's ability to interact with PIP2. This leads to chaotic actin bundles and a lack of focal adhesions in vinculin KO MEFs, which can be rescued by wild-type vinculin but not by the PIP2-binding mutant.
93
How can the interaction between talin and PIP2 be studied experimentally?
One method involves creating vesicles loaded with sucrose, with PIP2 incorporated on the outside. When these vesicles are centrifuged, proteins that interact with PIP2 will co-sediment with the vesicles, allowing for easy identification of PIP2-binding proteins such as talin.
94
What happens if PIP2 is absent in the experimental setup?
In the absence of PIP2, proteins like talin that rely on PIP2 for activation will fail to interact with the membrane, preventing integrin activation and focal adhesion formation.
95
How does PIP2 regulate the conformation of talin?
PIP2 binds to the FERM domain of talin, inducing a conformational change that allows talin to interact with integrins and actin. This interaction is essential for inside-out signaling, which activates integrins.
96
How does PIP2 affect vinculin?
The polybasic region (PBR) of vinculin interacts with PIP2, inducing a conformational change that activates vinculin. This active vinculin then recruits paxillin and strengthens the actin cytoskeleton-integrin interactions, contributing to the formation of large focal adhesions.
97
What is the role of PIP2 in the formation of focal adhesions?
PIP2 regulates the activation of talin and vinculin, two key proteins involved in focal adhesion formation. Activated talin binds to integrins, and activated vinculin recruits other proteins like paxillin, strengthening the link between integrins and the actin cytoskeleton to form focal adhesions.
98
How does PIP5Kγ isoform 1 localize to focal adhesions?
PIP5Kγ isoform 1 (661) localizes to focal adhesions through its interaction with talin. The integrin tail also binds to talin at a similar site, and PIP5Kγ competes with integrin for this interaction.
99
What are the isoforms of PIP5K, and how do they differ?
PIP5K has three isoforms: α, β, and γ. The γ isoform has four splice variants, with isoform 1 (661) being critical for localization at focal adhesions.
100
What are small molecular weight G-proteins (SMGs), and how are they activated?
SMGs (e.g., Rho, Rac, Cdc42) are monomeric G-proteins that are active when bound to GTP and inactive when GTP is hydrolyzed to GDP. They are regulated by guanine nucleotide exchange factors (GEFs), which activate them by promoting GTP binding, and GTPase activating proteins (GAPs), which deactivate them by hydrolyzing GTP.
101
How do SMGs interact with PIP5K?
SMGs bind to and recruit PIP5K, which plays a role in regulating the activity and localization of these G-proteins. PIP5K contributes to the generation of PIP2, which is involved in the activation cycle of SMGs.
102
What is the role of polybasic signaling in SMG regulation?
Many small molecular weight G-proteins (SMGs), such as RAS, RHO, ARF, and RAB, contain a lipid modification group and a polybasic region. This polybasic region allows these proteins to interact with membrane phosphoinositides like PIP2, which helps control their activation and localization.
103
How does PIP2 regulate Rac activation?
Rac, a G-protein, is lipid-modified and kept in its inactive GDP-bound state by guanine nucleotide dissociation inhibitor (GDI). Activation occurs through a guanine nucleotide exchange factor (GEF), which promotes the exchange of GDP for GTP. The polybasic region of Rac then interacts with phosphoinositides, including PIP2, leading to its activation.
104
How does PIP2 participate in a feed-forward mechanism to regulate Rac?
Rac activation leads to the activation of PIP5K, which produces PIP2. The generated PIP2 acts in a feed-forward manner to enhance GEF activity, promoting further activation of Rac. This cycle is important for the regulation of many SMGs and contributes to cellular signaling processes such as membrane dynamics and cytoskeletal rearrangements.
105
How is PIP2 regulated in specific cellular processes despite its widespread presence?
Specific PIP2 pools are generated at localized sites within the cell through specialized processes such as targeting by PIP5K interactors. This ensures that small changes in PIP2 concentration can be sensed at the site of action, even if the overall PIP2 level remains largely unchanged.
106
What is the role of PIP5K interactors in PIP2 regulation?
PIP5K interactors serve as targeting molecules that direct PIP5K to specific cellular domains, allowing the generation of localized pools of PIP2. These pools are then used by effectors that can act in response to changes in PIP2 concentration at these specific sites.
107
How are PIP2 changes sensed at the cellular level?
PIP2 changes are often sensed at nano domains that are beyond the resolution of light microscopy. These nano domains allow for specialized synthesis and regulation of PIP2 pools, ensuring that local changes in PIP2 are perceived differently than bulk PIP2 levels.
108
What are Flares in lipid dynamics, and how are they used?
Flares are fluorescent lipid-associated reporters that use different domains which specifically associate with lipids such as PIP2. These domains are genetically encoded as fusions with fluorescent proteins, allowing researchers to track changes in lipid levels dynamically in live cells by visualizing fluorescence.
109
How are different lipid domains tracked in live cells using Flares?
By expressing Flares in cells, different domains of lipid-binding proteins can be color-coded using various fluorescent proteins. This enables the real-time monitoring of lipid dynamics, including changes in PIP2 and other phosphoinositides.
110
How does PIP2 behave during phagocytosis?
During phagocytosis, PIP2 dramatically increases as actin polymerization occurs to form the phagocytic cup. As the bead is engulfed, PIP2 decreases, and PIP3 and DAG increase. Once the bead is internalized, PIP2 is no longer present, and actin depolymerizes.
111
What is the relationship between PIP2, PIP3, and DAG during phagocytosis?
As PIP2 decreases, PIP3 and DAG levels increase. The conversion of PIP2 to PIP3 through the activity of PI3K plays a crucial role in actin dynamics during phagocytosis.
112
How do PI3K inhibitors affect phagocytosis and PIP2?
PI3K inhibitors block the conversion of PIP2 to PIP3, preventing PIP2 depletion and actin polymerization, which ultimately inhibits phagocytosis. This highlights the role of PIP2 in regulating actin polymerization during the engulfment process.
113
What is the role of PIP5K in phagocytosis?
PIP5K is recruited to the phagocytic cup, likely through activation by Fc receptors. This recruitment facilitates the localized generation of PIP2 at the site of phagocytosis, contributing to actin polymerization and membrane remodeling.
114
How does PIP5K localization change during phagocytosis?
During phagocytosis, PIP5K is recruited to the phagocytic cup and then dissociates as the cup internalizes the bead. The change in localization is essential for regulating PIP2 levels at the site of the phagosome formation.
115
How is PIP2 generated and utilized in specific cellular domains?
PIP2 is generated by PIP5K, which is targeted to specific cellular domains by interactors. These interactors ensure that PIP2 is produced locally, where it can then be used by effectors to regulate processes like actin polymerization, membrane dynamics, and signaling.
116
How do PIP5K interactors contribute to PIP2 regulation?
PIP5K interactors are responsible for targeting PIP5K to specific locations in the cell, enabling the local production of PIP2. The localized PIP2 pool can then be sensed and used by effector proteins, ensuring precise regulation of cellular processes like phagocytosis and membrane trafficking.
117
What is the role of PIP2 in clathrin-coated pit formation?
PIP2 is essential for the clustering of clathrin adaptor proteins (CAPs) such as AP2, AP180, and Epsin. These proteins bind to PIP2 and the coat protein clathrin, helping to form and stabilize the curved membrane during endocytosis.
118
How do clathrin adaptor proteins (CAPs) interact with PIP2 during clathrin-coated pit formation?
CAPs like AP2, AP180, and Epsin have domains that specifically bind PIP2 (e.g., the ANTH domain in AP180 and ENTH domain in Epsin). This interaction helps the clustering of adaptor proteins and clathrin, which contributes to membrane deformation and pit formation.
119
What is the role of dynamin in the formation of clathrin-coated pits?
Dynamin, a GTPase, is responsible for the scission (pinching off) of the clathrin-coated vesicle from the membrane. It binds to PIP2 via its PH domain, and GTP hydrolysis provides the energy required for the vesicle's membrane scission.
120
How does PIP2 contribute to dynamin activity in clathrin-mediated endocytosis?
PIP2 interacts with the PH domain of dynamin, which recruits dynamin to the vesicle neck. This is crucial for membrane pinching and vesicle scission. The GTP hydrolysis by dynamin is believed to drive the scission process.
121
What role does Endophilin play in clathrin-coated vesicle formation and scission?
Endophilin senses membrane curvature through its BAR domain and binds PIP2 to further induce curvature. It also recruits Synaptojanin 2, a PIP2 5-phosphatase that degrades PIP2 specifically in the vesicle, triggering uncoating and the completion of vesicle formation.
122
What is the role of Synaptojanin 2 in clathrin-coated vesicle uncoating?
Synaptojanin 2 is recruited by Endophilin to the vesicle, where it acts as a PIP2 5-phosphatase, removing PIP2 from the vesicle membrane. This process is critical for the uncoating of the vesicle after it buds from the membrane.
123
How does PIP2 influence vesicle budding at the Trans-Golgi Network (TGN)?
Similar to clathrin-mediated endocytosis at the plasma membrane, PIP2 is required for vesicle budding at the TGN. This process involves the clathrin adaptor protein AP1, and PIP2 synthesis is also necessary at this site to facilitate proper vesicle formation.
124
How is PIP2 involved in both plasma membrane and Trans-Golgi Network (TGN) vesicle formation?
PIP2 plays a role in vesicle budding at both the plasma membrane and the TGN. At the plasma membrane, it helps with clathrin-coated pit formation, while at the TGN, it aids in vesicle formation through interaction with AP1 and other clathrin adaptors.
125
What is the function of the endosome in cellular trafficking?
The endosome is a key sorting organelle in the cell. It receives vesicles from the plasma membrane (PM) or Golgi, and the cargo within these vesicles can either be recycled back to the PM or degraded by fusion with lysosomes.
126
How do vesicles from the plasma membrane (PM) interact with the endosome?
Vesicles from the PM or Golgi fuse with the early endosome. The cargo within these vesicles can be either recycled back to the PM or undergo maturation into late endosomes, which then fuse with lysosomes for degradation.
127
How does PIP2 contribute to phagosome maturation?
During the early phase of phagosome maturation, PIP2 is initially present on the phagosome membrane but is later replaced by PI3P. This conversion is crucial for the phagosome's entry into the endocytic system, where it can eventually fuse with early endosomes.
128
How does the generation of PI3P influence phagosome maturation?
PI3P is generated after PIP2 is hydrolyzed during phagosome maturation. PI3P helps recruit the necessary machinery for the phagosome to fuse with the early endosome, facilitating the progression of the phagocytic process.
129
What role does PIP2 regulation play in neurodegenerative diseases?
Deregulation of Synaptojanin 1, a PIP2-phosphatase, is implicated in various neurodegenerative diseases such as Down syndrome, early-onset Parkinson's disease, and Alzheimer's disease. These conditions may involve dysfunction in the PIP2-mediated processes of synaptic vesicle uncoating and trafficking.
130
What is the role of PI3P in the endocytic system?
PI3P is a critical lipid within the endocytic system. It is involved in various processes such as vesicle trafficking, maturation, and fusion of early endosomes, and regulates subcellular compartment identity through dynamic changes in phosphoinositides.
131
How can the modulation of PI3P influence cellular activities?
PI3P can be modulated to drive different cellular outcomes by coordinating with specific domains, which allows for coincidence detection and functional regulation of subcellular compartments.
132
What is coincidence detection and how does it influence PI3P signaling?
Coincidence detection refers to the simultaneous interaction of PI3P with other specific membrane-targeting domains (e.g., PX or FYVE domains). This low-affinity interaction, when combined with another targeting domain, enhances avidity and drives specific cellular outcomes, enabling PI3P to regulate different processes.
133
Why is coincidence detection important for PI3P?
Coincidence detection allows PI3P to drive distinct functions by combining its low-affinity interactions with other domains that act as stabilizers and enhancers of specific membrane targeting. This increases the specificity of PI3P in regulating different cellular functions.
134
What is the role of the FYVE domain in PI3P detection?
The FYVE domain is a PI3P binding domain that coordinates 2 zinc atoms and has a hydrophobic loop that helps insert into the membrane, stabilizing the interaction with PI3P. This domain binds primarily to PI3P, with some interaction also with PI(3,4)P2, and has low affinity. There are 87 FYVE-containing proteins in humans.
135
How does the PX domain contribute to PI3P binding and detection?
The PX domain binds PI3P through a hydrophobic loop and a pocket formed by beta-sheets and alpha-helices. It has variable affinity for PI3P, and can also interact with PI(3,4)P2 and PI(4,5)P2. There are 108 PX-containing proteins in humans, many of which are involved in the endosomal pathway.
136
How does PI3P function in the maturation of endosomes?
In the early endosome, PI3P is important for vesicle conversion and maturation. The interaction between RAB7 and PI3P on vesicles helps identify more mature endosomal compartments. LC3 binds RAB7/PI3P vesicles, contributing to autophagy and targeting vesicles to lysosomes for degradation.
137
How does PI3P and the FYVE domain contribute to endosomal maturation and degradation of ubiquitinated proteins?
The protein HRS1, containing a FYVE domain and a ubiquitin interacting motif (UIM), detects PI3P and ubiquitinated proteins. This drives the maturation of late endosomes and facilitates the degradation of proteins, such as the EGF receptor, through the ESCRT complex.
138
How does PI3P contribute to retromer-mediated recycling?
The SNX1 protein contains a PX domain that binds to PI3P and a BAR domain that senses membrane curvature. Coincidence detection of PI3P and membrane curvature drives tubulation and trafficking of endosomes back to the Trans-Golgi Network (TGN) for recycling.
139
What is the role of PI3P and membrane curvature in endosome-to-TGN trafficking?
The retromer complex recognizes PI3P and membrane curvature to drive the endosome-to-TGN trafficking process. PI3P in combination with specific types of membrane curvature promotes the formation of tubular structures that facilitate cargo recycling back to the TGN.
140
What is the functional significance of PI3P in autophagy?
PI3P helps to regulate autophagy by associating with RAB7-containing vesicles and recruiting LC3, a protein involved in autophagosome formation. This interaction facilitates the transport of vesicles to lysosomes for degradation.
141
How is PI3P involved in cellular sorting and degradation pathways?
PI3P plays a key role in the sorting and degradation of proteins by coordinating with the ESCRT complex to mediate membrane scission. This facilitates the degradation of ubiquitinated proteins, such as the EGF receptor, within the late endosome or lysosome.
142
What is the BAR domain and its role in membrane curvature sensing?
The BAR domain (Bin-Amphiphysin-Rvs) is a highly conserved protein dimerization domain that is banana-shaped, allowing it to interact with and sense membrane curvature. It plays a key role in membrane deformation and the initiation of processes such as vesicle formation.
143
How do N-BAR domains differ from the standard BAR domain?
N-BAR domains contain an alpha-helix preceding the BAR domain, which enhances their ability to mediate membrane tubulation. These are often found in conjunction with PX domains in the SNX family of proteins.
144
What is the key function of the BAR domain family of proteins?f
The BAR domain family of proteins is involved in membrane bending and binding. These proteins play a crucial role in inducing membrane curvature and stabilizing the structures necessary for vesicle formation and trafficking.
145
What is coincidence detection in lipid signaling?
Coincidence detection occurs when proteins with multiple functional domains bind to different lipid domains, leading to specific signaling outputs. This mechanism enables specificity in cellular processes by linking proteins to distinct lipid environments, where each combination can trigger different cellular outcomes.
146
How does dimerization of proteins, like dynamin, contribute to lipid signaling?
Dimerization increases the affinity of proteins for specific lipids. For example, dynamin doesn’t bind PIP2 well in its monomeric form, but upon oligomerization, it gains more binding sites, enabling it to interact more effectively with the lipid membrane, promoting processes like membrane scission.
147
How do weak interactions between proteins and lipids enhance specificity in cellular signaling?
Weak interactions, such as those between proteins and lipids, allow for flexibility and specificity in how proteins can interact with different lipid microenvironments. This coupling enables proteins to engage with multiple lipids at once, which helps generate distinct cellular outcomes based on the lipid composition and context.
148
How do PI cascades regulate cellular outputs in a coordinated manner?
PI cascades involve the sequential phosphorylation and dephosphorylation of phosphoinositides, which regulate cellular processes like vesicle trafficking, membrane dynamics, and signal transduction. The interactions between phosphoinositides and various proteins can generate different outputs depending on the specific protein partners involved.
149
How do kinases and phosphatases contribute to the regulation of PI cascades?
Kinases and phosphatases control the synthesis and degradation of specific phosphoinositides, which modulate their functional interactions with various effector proteins. These enzymes enable dynamic regulation of cellular processes by altering the availability of specific lipid species at distinct membrane microdomains.
150
How can the same phosphoinositide drive different cellular outcomes?
The same phosphoinositide can drive different outcomes based on the proteins it associates with. Different lipid-binding domains (such as PX or FYVE) and functional protein domains (such as kinase or phosphatase domains) enable specific interactions, leading to varied signaling pathways and cellular responses depending on the protein context.
151
How do PI cascades influence cellular compartment functions?
PI cascades modulate the identity and function of subcellular compartments by regulating the dynamics of phosphoinositides within specific membranes. This, in turn, influences processes like endocytosis, exocytosis, vesicle trafficking, and membrane remodeling, all of which require precise lipid-protein interactions.
152
How does coincidence detection contribute to membrane trafficking and organelle function?
Coincidence detection allows for precise recruitment of proteins to specific lipid domains, such as PI3P in early endosomes or PIP2 in clathrin-coated pits. By coupling lipid binding with functional domains, proteins can perform specific roles in membrane trafficking, organelle maturation, and vesicle fusion or scission.
153
What is the role of the BAR domain in vesicle formation?
The BAR domain helps mediate the formation of membrane tubules by sensing and stabilizing the curvature of the membrane. It can bind to the membrane and induce curvature necessary for the formation of clathrin-coated pits and other vesicle types, playing a central role in membrane remodeling.
154
How do BAR domains and N-BAR domains work together in the SNX family?
BAR domains and N-BAR domains in the SNX family function in membrane tubulation and trafficking. While BAR domains contribute to membrane curvature sensing, the alpha-helix in N-BAR domains enhances the ability to induce tubulation, working synergistically with other domains like PX to direct vesicle formation and trafficking.
