3. Cell Division and Cell Division Cell Plane Orientation Flashcards
What marks the cell division zone (CDZ) from prophase to cytokinesis?
An actin-free zone at the cell cortex
How does sabre and clasp mutants behave?
CLASP is a microtubule (MT)-associated protein involved in attachment of MTs to the cortex in animals and plants. It regulates the dynamics of the MT plus ends. SABRE (SAB) is a large protein of unknown molecular function required for proper MT alignment. sab mutations act genetically epistatically to clasp mutants in Arabidopsis regarding PPB orientation, spindle and phragmoplast orientation.
sabre mutants display defective PPB and cell division plane orientation
How are tonneau1 (ton1) and tonneau2/fass good for?
They are required for formation and/or the integrity of the PPB
What have the ton1, ton2/fass and protein phosphatase2a mutants have in common?
The display a missorientation in cell division during early embryogenesis. They are all associated with the PPB.
Arabidopsis TON1a and TON1b are almost identical. The Arabidopsis TON1 show homology with which proteins?
To animal, centrosomal proteins bind to centrins and localises to the PPB.
How does the method of “tandem affinity purification” also “TAP tagging” works? It’s a specific immunoprecipitation method.
- The GS-Tap-TAG consists of the protein G (G) and streptavidin-binding protein (SBP), fused to the target protein e.g. TON2 and the fusion is expressed in plant cell suspension cultures.
SBP: kann ziehen oder lockern, je nachdem ob Streptavidin gebunden ist oder nicht.
GS Protein G – Protein G- TEV-SBP+ bait protein
Proteing-Protein G-TEV binds to IGG; TEV is a protein clevage side; protease cleaves out the whole complex; Pulling down complex binds to IGG - GS-TAP-TON2/TON1 is incubated with the protein extract and than incubated with IgG sepharose beads (?), Subsequently, the beads can be centrifuged down and the protein complex can be precipitated. It is washed and incubated with TEV protease which releases the protein complex
- The complex is incubated wuth streptavidin-loaded sepharose beads. Those are centrifuged down to precipitate the protein complex. Afterwards, they are washed and the streptavidin is outcompeted by biotin releasing the purified complex subsequently: fragmentation of the proteins and peptide identification by mass spectrometry
In TAP-tag experiments, it has been shown that TON1 and CDKA;1 coprecipitate – what has not been shown right now?
If CDKA;1 regulates the activity of TON1
TAN, RanGAP1/2, POK1/2: Who interacts with who and what do you know about these 3 players?
TANGLED (TAN) and RanGAP1/2 proteins remain at the cortical cell division zone (CDZ) during mitosis and cytokinesis!
The phragmoplast-orienting kinesins (POK) POK1 and POK2 can interact directly with Arabidopsis TANGLED and are used for the correct orientation of the phragmoplast as well as the cell division plane.
What had been identified by TAP tagging?
The new adapter complex: The TPLATE complex of clathrin dependent endocytosis was identified by TAP tagging of TPLATE and other components.
What do you know about the T-plate?
It is localisied at cell division plane and membrane (where the cell plate occurs) and looks like a “T”
Name some cargo molecules of the “TPLATE -Komplex”!
PIN1 and PIN2: are polarly localized auxin efflux carriers
BRI1: a brassinosteroid receptor
KNOLLE: a cytokinesis-specific syntaxin
Name some adaptor components of the TPLATE complex!
AtEH1 and AtEH2 are epsin-homology-domain proteins.
LOLITA
TML is another key component
TPLATE is a plant specific componten, it delivers compontens back
TMW40-1
TMW40-2
AP2 complex is an clathrin adapter complex required for endocytosis from the plasma membrane
For cell expansion – phragmoplast has to be stable or instable? Explain!
Phragmoplast instability at the midzone
If MAP65 is not phosphorylated: stable phragmoplast
Phragmoplast in late anaphase
MAP65 phosphorylated by AtMPK4 = phragmoplast instability in the midzone –> MT turneover –> phragmoplast expansion
