11/9
Disassembly of Actin and Keratin Networks by Aurora B Kinase at the Midplane of Cleaving Xenopus laevis Eggs (Current Biology 2019 Tim Mitchison)
Aurora B kinase activity between asters disassembles actin and keratin networks
Cytoskeleton disassembly results in reduced cytoplasmic stiffness between asters
Aurora B kinase bound to beads causes local disassembly of cytoskeleton networks
Microtubule binding locally amplifies Aurora B kinase activity
CPC - mitotic kinase Aurora B / three additional subunits (INCENP, Survivin, and Borealin) that localize and activate the kinase
Centralspindlin - kinesin-6, MKLP1, and two molecules of MgcRacGAP, which contains a GTPase-activating protein (GAP) domain for Rho family GTPases.
CPC - the boundary between asters.
Aurora B kinase (AURKB) (subunit of the CPC) - caused disassembly of F-actin and keratin between asters
cytokinesis signaling protein complexes: chromosomal passenger complex (CPC) and Centralspindlin
A requirement for CPC activity for cytokinesis was shown in several large cell types, including zebrafish [4], sea urchin [5], and Xenopus [6].
CPC is activated by auto-phosphorylation, which is enhanced by binding to microtubules [7]
a CPC-positive state from anaphase chromatin to the egg cortex is spread via microtubule bundles between asters [1][2]
F-actin and keratin networks - impede centrosome movement and furrow ingression
A plausible model is that CDK1 promotes global network disassembly at mitotic entry, whereas AURKB adds a focused disassembly activity that prevents re-formation at the midplane, which might impede furrow ingression.
CDK1
Actin-intact interphase egg extract containing fluorescent probes was spread between passivated coverslips and imaged.
AURKB and Prc1/Kif4A activities together block plus-end growth and generate sharp boundaries between asters, as previously reported [6, 19]
an AURKB kinase inhibitor - inhibit actin’s disassembly
Keratin networks were also disrupted at aster boundaries when CPC was recruited (slower than f-actin)
To examine these phenomena in vivo, we analyzed intact zygotes using fixation, clearing, and laser-scanning confocal imaging
The mechanism of local disassembly of F-actin by AURKB is not known.
How AURKB destabilizes keratin filaments is also unknown
Disassembly of Actin and Keratin Networks by Aurora B Kinase at the Midplane of Cleaving Xenopus laevis Eggs (Current Biology 2019 Tim Mitchison)
Aurora B kinase activity between asters disassembles actin and keratin networks
Cytoskeleton disassembly results in reduced cytoplasmic stiffness between asters
Aurora B kinase bound to beads causes local disassembly of cytoskeleton networks
Microtubule binding locally amplifies Aurora B kinase activity
CPC - mitotic kinase Aurora B / three additional subunits (INCENP, Survivin, and Borealin) that localize and activate the kinase
Centralspindlin - kinesin-6, MKLP1, and two molecules of MgcRacGAP, which contains a GTPase-activating protein (GAP) domain for Rho family GTPases.
CPC - the boundary between asters.
Aurora B kinase (AURKB) (subunit of the CPC) - caused disassembly of F-actin and keratin between asters
cytokinesis signaling protein complexes: chromosomal passenger complex (CPC) and Centralspindlin
A requirement for CPC activity for cytokinesis was shown in several large cell types, including zebrafish [4], sea urchin [5], and Xenopus [6].
CPC is activated by auto-phosphorylation, which is enhanced by binding to microtubules [7]
a CPC-positive state from anaphase chromatin to the egg cortex is spread via microtubule bundles between asters [1][2]
F-actin and keratin networks - impede centrosome movement and furrow ingression
A plausible model is that CDK1 promotes global network disassembly at mitotic entry, whereas AURKB adds a focused disassembly activity that prevents re-formation at the midplane, which might impede furrow ingression.
CDK1
Actin-intact interphase egg extract containing fluorescent probes was spread between passivated coverslips and imaged.
AURKB and Prc1/Kif4A activities together block plus-end growth and generate sharp boundaries between asters, as previously reported [6, 19]
an AURKB kinase inhibitor - inhibit actin’s disassembly
Keratin networks were also disrupted at aster boundaries when CPC was recruited (slower than f-actin)
To examine these phenomena in vivo, we analyzed intact zygotes using fixation, clearing, and laser-scanning confocal imaging
The mechanism of local disassembly of F-actin by AURKB is not known.
How AURKB destabilizes keratin filaments is also unknown
11/9
Spatial Organization of Cytokinesis Signaling Reconstituted in a Cell-Free System (Science 2014 Tim Mitchison)
The CPC was transported to overlap zones, which required two motor proteins, Kif4A and a Kif20A paralog.
cleavage furrow markers (including a RhoA-GTP reporter) was observed to be recruited to microtubule overlaps
the signaling complexes Centralspindlin and CPC (Chromosomal Passenger Complex) accumulate at the center of the midzone (or central spindle), which forms in the space previously occupied by the mitotic spindle
Where the expanding edges of two neighboring asters met, antiparallel microtubule bundles formed in a boundary zone that we term the aster-aster interaction zone (AAIZ)
CPC and Centralspindlin complexes are recruited to the midplane in anaphase - activate the small GTPase RhoA - to specify the division plane
CPC - is labeled by a GFP tagged DasraA subunit
CPC (AurkB) and the Kif23 subunit of Centralspindlin were recruited to the AAIZ
AAIZs in the cell-free system can signal to the cortex
The small GTPase RhoA is thought to be the master organizer of the furrow (16)
To localize active, GTP-bound RhoA we added an mCherry-tagged RhoA binding fragment of Rhotekin (mCherry-rGBD) (16).
the RhoA-GTP reporter was enriched at the bilayer under AAIZs that recruited CPC and Centralspindlin
a CPC-positive AAIZ can locally activate RhoA in our system
But we cannot distinguish whether localized AurkB phosphorylation of cytokinetic factors or some specific organization of microtubules at the AAIZ is required for furrow signaling.
Spatial Organization of Cytokinesis Signaling Reconstituted in a Cell-Free System (Science 2014 Tim Mitchison)
The CPC was transported to overlap zones, which required two motor proteins, Kif4A and a Kif20A paralog.
cleavage furrow markers (including a RhoA-GTP reporter) was observed to be recruited to microtubule overlaps
the signaling complexes Centralspindlin and CPC (Chromosomal Passenger Complex) accumulate at the center of the midzone (or central spindle), which forms in the space previously occupied by the mitotic spindle
Where the expanding edges of two neighboring asters met, antiparallel microtubule bundles formed in a boundary zone that we term the aster-aster interaction zone (AAIZ)
CPC and Centralspindlin complexes are recruited to the midplane in anaphase - activate the small GTPase RhoA - to specify the division plane
CPC - is labeled by a GFP tagged DasraA subunit
CPC (AurkB) and the Kif23 subunit of Centralspindlin were recruited to the AAIZ
AAIZs in the cell-free system can signal to the cortex
The small GTPase RhoA is thought to be the master organizer of the furrow (16)
To localize active, GTP-bound RhoA we added an mCherry-tagged RhoA binding fragment of Rhotekin (mCherry-rGBD) (16).
the RhoA-GTP reporter was enriched at the bilayer under AAIZs that recruited CPC and Centralspindlin
a CPC-positive AAIZ can locally activate RhoA in our system
But we cannot distinguish whether localized AurkB phosphorylation of cytokinetic factors or some specific organization of microtubules at the AAIZ is required for furrow signaling.
11/9
A microtubule-dependent zone of active RhoA during cleavage plane specification (JCB 2005 George von Dassow)
The small GTPase RhoA has previously been implicated in cytokinesis.
They observed that active RhoA concentrates in a precisely bounded zone before cytokinesis and is independent of actin assembly.
microtubules specify the cytokinetic apparatus via a dynamic zone of local RhoA activity.
A microtubule-dependent zone of active RhoA during cleavage plane specification (JCB 2005 George von Dassow)
The small GTPase RhoA has previously been implicated in cytokinesis.
They observed that active RhoA concentrates in a precisely bounded zone before cytokinesis and is independent of actin assembly.
microtubules specify the cytokinetic apparatus via a dynamic zone of local RhoA activity.
11/21
Rho GTPases in animal cell cytokinesis: An occupation by the one percent
Rho GTPases in animal cell cytokinesis: An occupation by the one percent
12/2
RNA-triggered protein cleavage and cell growth arrest by the type III-E CRISPR nuclease-protease
RNA-activated protein cleavage with a CRISPR-associated endopeptidas (Feng Zhang)
The Cas7-11–Csx29 effector is an RNA-dependent nuclease-protease system and is an important part of the bacterial anti-phage immune system. The system performs calculations at the post-translational protein level, so it has the potential to detect cancer cells and specific cell subtypes.
RNA-triggered protein cleavage and cell growth arrest by the type III-E CRISPR nuclease-protease
RNA-activated protein cleavage with a CRISPR-associated endopeptidas (Feng Zhang)
The Cas7-11–Csx29 effector is an RNA-dependent nuclease-protease system and is an important part of the bacterial anti-phage immune system. The system performs calculations at the post-translational protein level, so it has the potential to detect cancer cells and specific cell subtypes.
12/2
Structural basis for Cas9 off-target activity (Cell 2022 Switzerland Martin Jinek)
Cas9: complement guide CRISPR RNA (crRNA) by cleaving double-stranded DNA (dsDNA) substrates. In addition, by changing the sequence of the guide RNA (gRNA), the DNA-specific programming of the CRISPR-Cas9 system can also be easily realized.
This paper reports the crystal structure of Cas9 binding to a bona fide off-target substrate, revealing that off-target binding is achieved by a series of non-canonical base-pairing interactions in the heteroduplex directing the off-target
These studies provide a structural basis for the off-target activity of Cas9 and help improve the rational design of guide RNAs and off-target prediction algorithms.
Structural basis for Cas9 off-target activity (Cell 2022 Switzerland Martin Jinek)
Cas9: complement guide CRISPR RNA (crRNA) by cleaving double-stranded DNA (dsDNA) substrates. In addition, by changing the sequence of the guide RNA (gRNA), the DNA-specific programming of the CRISPR-Cas9 system can also be easily realized.
This paper reports the crystal structure of Cas9 binding to a bona fide off-target substrate, revealing that off-target binding is achieved by a series of non-canonical base-pairing interactions in the heteroduplex directing the off-target
These studies provide a structural basis for the off-target activity of Cas9 and help improve the rational design of guide RNAs and off-target prediction algorithms.
12/2
Refactored genetic codes enable bidirectional genetic isolation (Science 2022 Jason W. Chin Cambridge university)
Horizontal gene transfer (HGT): one way for evolution - The process of passing genetic material to other cells rather than progeny, such as conjugation, transduction, and transformation of cells
Gene vertical transfer: the process by which organisms pass genes to offspring.
This article reconstructs the genetic code structure of Escherichia coli and creates an orthogonal genetic code that limits the escape of synthetic genetic information into natural organisms.
A new codon rule was defined in which the TCG and TCA serine codons were replaced with the synonymous AGC and AGT codons, and the TAG stop codon was also replaced with TAA, resulting in the Syn61 strain - further improve it to Syn61∆3: Can barely read all codons in the universal genetic code
Refactored genetic codes enable bidirectional genetic isolation (Science 2022 Jason W. Chin Cambridge university)
Horizontal gene transfer (HGT): one way for evolution - The process of passing genetic material to other cells rather than progeny, such as conjugation, transduction, and transformation of cells
Gene vertical transfer: the process by which organisms pass genes to offspring.
This article reconstructs the genetic code structure of Escherichia coli and creates an orthogonal genetic code that limits the escape of synthetic genetic information into natural organisms.
A new codon rule was defined in which the TCG and TCA serine codons were replaced with the synonymous AGC and AGT codons, and the TAG stop codon was also replaced with TAA, resulting in the Syn61 strain - further improve it to Syn61∆3: Can barely read all codons in the universal genetic code
12/2
A cytokinesis furrow is positioned by two consecutive signals (Nature 2005 MPI)
Spindle midzone or aster - determine the cleavage furrow site
the cytokinesis furrow is first positioned by a signal determined by microtubule asters, and then by a second signal that is derived from the spindle midzone
A cytokinesis furrow is positioned by two consecutive signals (Nature 2005 MPI)
Spindle midzone or aster - determine the cleavage furrow site
the cytokinesis furrow is first positioned by a signal determined by microtubule asters, and then by a second signal that is derived from the spindle midzone
12/3
The incidence of cytoplasmic fragmentation in mouse embryos in vitro is not affected by inhibition of caspase activity (Fertility and sterility 2021 HKU)
Caspases are a family of endoproteases that provide critical links in cell regulatory networks controlling inflammation and cell death.
Caspases are a family of cytosolic aspartate-specific cysteine proteases involved in the initiation and execution of apoptosis
the formation of fragments in precompaction mouse embryos is independent of caspase activity and that embryo fragments are not apoptotic bodies.
The incidence of cytoplasmic fragmentation in mouse embryos in vitro is not affected by inhibition of caspase activity (Fertility and sterility 2021 HKU)
Caspases are a family of endoproteases that provide critical links in cell regulatory networks controlling inflammation and cell death.
Caspases are a family of cytosolic aspartate-specific cysteine proteases involved in the initiation and execution of apoptosis
the formation of fragments in precompaction mouse embryos is independent of caspase activity and that embryo fragments are not apoptotic bodies.
12/3
Cytoplasmic fragmentation in activated eggs occurs in the cytokinetic phase of the cell cycle, in lieu of normal cytokinesis, and in response to cytoskeletal disorder (Molecular Human Reproduction 2005 Australia)
fragmentation does not occur in mitotically inactive cells
fragmentation in oocytes and embryos occurs during the cytokinetic phase of the cell cycle, in response to the loss of normal interplay between the spindle complex and cortical microfilaments
fragmentation in oocytes and early embryos, though seemingly uncoordinated, is a precisely timed event that occurs only in mitotically active cells, during the cytokinetic phase of the cell cycle, in lieu of normal cytokinesis, and in response to altered cytoskeletal organization.
Cytoplasmic fragmentation in activated eggs occurs in the cytokinetic phase of the cell cycle, in lieu of normal cytokinesis, and in response to cytoskeletal disorder (Molecular Human Reproduction 2005 Australia)
fragmentation does not occur in mitotically inactive cells
fragmentation in oocytes and embryos occurs during the cytokinetic phase of the cell cycle, in response to the loss of normal interplay between the spindle complex and cortical microfilaments
fragmentation in oocytes and early embryos, though seemingly uncoordinated, is a precisely timed event that occurs only in mitotically active cells, during the cytokinetic phase of the cell cycle, in lieu of normal cytokinesis, and in response to altered cytoskeletal organization.
12/7
Rho GTPases in animal cell cytokinesis: An occupation by the one percent (Cytoskeleton (Hoboken) 2012)
Rac1 is a negative regulator of cytokinesis
inactivated within the division plane
actived in the cell poles
Cdc42 is also required
RhoA - binds to GTP (active); binds to GDP (inactive)
RhoA localizes to equatorial membrane before cell division occurs
RhoA protein accumulates at the division plane before the cell begins to divide
It is hypothesized that spindle microtubules dictate the site of cell division by specifying RhoA activation. Indeed, following microtubule depolymerization, GTP-bound RhoA does not properly localize to the cell equator
Rho GTPases in animal cell cytokinesis: An occupation by the one percent (Cytoskeleton (Hoboken) 2012)
Rac1 is a negative regulator of cytokinesis
inactivated within the division plane
actived in the cell poles
Cdc42 is also required
RhoA - binds to GTP (active); binds to GDP (inactive)
RhoA localizes to equatorial membrane before cell division occurs
RhoA protein accumulates at the division plane before the cell begins to divide
It is hypothesized that spindle microtubules dictate the site of cell division by specifying RhoA activation. Indeed, following microtubule depolymerization, GTP-bound RhoA does not properly localize to the cell equator
12/7
Spindle Assembly in Xenopus Egg Extracts: Respective Roles of Centrosomes and Microtubule Self-Organization (JCB 1997 Rebecca Heald)
Centrosomes provide dominant sites for pole formation
But it’s not required in spindle assembly - without pole, microtubules can still be sorted in an anti-parallel way
Spindle Assembly in Xenopus Egg Extracts: Respective Roles of Centrosomes and Microtubule Self-Organization (JCB 1997 Rebecca Heald)
Centrosomes provide dominant sites for pole formation
But it’s not required in spindle assembly - without pole, microtubules can still be sorted in an anti-parallel way
12/9
Preparation of Cellular Extracts from Xenopus Eggs and Embryos (CSH protocol 2018 Rebecca Heald)
Preparation of Cellular Extracts from Xenopus Eggs and Embryos (CSH protocol 2018 Rebecca Heald)
12/9
Isolation and Demembranation of Xenopus Sperm Nuclei (CSH protocol 2018 James Hazel)
Isolation and Demembranation of Xenopus Sperm Nuclei (CSH protocol 2018 James Hazel)
12/12
A printable active network actuator built from an engineered biomolecular motor (Nature materials 2021 Yuichi Hiratsuka Japan Advanced Institute of Science and Technology)
an active network capable of macroscopic actuation that is hierarchically assembled from an engineered kinesin, a biomolecular motor, and microtubules, resembling the contractile units in muscles
These contracting materials can be formed in desired areas using patterned ultraviolet illumination, allowing their incorporation into mechanically engineered systems, being also compatible with printing technologies.
A printable active network actuator built from an engineered biomolecular motor (Nature materials 2021 Yuichi Hiratsuka Japan Advanced Institute of Science and Technology)
an active network capable of macroscopic actuation that is hierarchically assembled from an engineered kinesin, a biomolecular motor, and microtubules, resembling the contractile units in muscles
These contracting materials can be formed in desired areas using patterned ultraviolet illumination, allowing their incorporation into mechanically engineered systems, being also compatible with printing technologies.
12/12
Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification (JCB 2003 Tim Mitchison)
Microtubules (MTs) polymerized with GMPCPP, a slowly hydrolyzable GTP analogue, are stable in buffer but are rapidly depolymerized in Xenopus egg extracts. (this depolymerization is independent of three previously identified MT destabilizers (Op18, katanin, and XKCM1/KinI)
XMAP215 is identified as the novel depolymerization factor
They suggest that CPP MTs are a model for the MT pause state - an obligate intermediate between polymerization and depolymerization
XMAP215 destabilizes the pause state, acting as an antipause factor
Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification (JCB 2003 Tim Mitchison)
Microtubules (MTs) polymerized with GMPCPP, a slowly hydrolyzable GTP analogue, are stable in buffer but are rapidly depolymerized in Xenopus egg extracts. (this depolymerization is independent of three previously identified MT destabilizers (Op18, katanin, and XKCM1/KinI)
XMAP215 is identified as the novel depolymerization factor
They suggest that CPP MTs are a model for the MT pause state - an obligate intermediate between polymerization and depolymerization
XMAP215 destabilizes the pause state, acting as an antipause factor
12/12
Midblastula transition (MBT) of the cell cycles in the yolk and pigment granule-free translucent blastomeres obtained from centrifuged Xenopus embryos (development, growth, & differentiation Yasuhiro Iwao Yamaguchi University)
We obtained translucent blastomeres free of yolk and pigment granules from Xenopus embryos which had been centrifuged at the beginning of the 8-cell stage with cellular integrity
Midblastula transition (MBT) of the cell cycles in the yolk and pigment granule-free translucent blastomeres obtained from centrifuged Xenopus embryos (development, growth, & differentiation Yasuhiro Iwao Yamaguchi University)
We obtained translucent blastomeres free of yolk and pigment granules from Xenopus embryos which had been centrifuged at the beginning of the 8-cell stage with cellular integrity
12/12
Cellular and Molecular Nature of Fragmentation of Human Embryos (2022 International Journal of Molecular Sciences Italy)
fragmentation can affect none, some, or all embryos, demonstrating that this phenomenon is both embryo- and patient-specific
A consistent body of literature linked the rate of fragmentation with a lower embryo developmental potential and implantation rate - This observation could be explained by the theory supporting an association between fragment-like entities and chromosomal abnormalities. Aberrant or exceeded chromosomes may result in the formation of DNA-containing micronuclei that ultimately may lead to the inactivation of the embryonic genome and the subsequent block of embryo development
On the other hand, a reduced embryo developmental potential may be explained also by those theories sup- porting the anucleate cytoplasmic nature of embryo cellular fragments. The loss of a large volume of cytoplasm may be detrimental to embryo potential by depleting blastocyst of essential organelles (e.g., mitochondria), mRNAs, and proteins, resulting in an early block during embryo development.
In addition, the presence of large cytoplasmic cellular fragments may also have an impact in the spatial arrangement of the blastomere in the context of ICM and trophectoderm of the blastocyst. Indeed, they can cause apoptosis or the loss of a significant volume of cytoplasm, limiting the rate of blastomere cleavage because of the distortion of the blastomere division planes, leading to abnormal compaction, cavitation and blastocyst formation.
cosmetic embryo microsurgery in terms of removal of fragments and coarse granulation from the embryo before embryo transfer has been suggested to improve cell division and implantation potential
cellular fragments could be indicative of chromosomal problems, and therefore their removal might not improve the potential of many severely fragmented embryos.
Cellular and Molecular Nature of Fragmentation of Human Embryos (2022 International Journal of Molecular Sciences Italy)
fragmentation can affect none, some, or all embryos, demonstrating that this phenomenon is both embryo- and patient-specific
A consistent body of literature linked the rate of fragmentation with a lower embryo developmental potential and implantation rate - This observation could be explained by the theory supporting an association between fragment-like entities and chromosomal abnormalities. Aberrant or exceeded chromosomes may result in the formation of DNA-containing micronuclei that ultimately may lead to the inactivation of the embryonic genome and the subsequent block of embryo development
On the other hand, a reduced embryo developmental potential may be explained also by those theories sup- porting the anucleate cytoplasmic nature of embryo cellular fragments. The loss of a large volume of cytoplasm may be detrimental to embryo potential by depleting blastocyst of essential organelles (e.g., mitochondria), mRNAs, and proteins, resulting in an early block during embryo development.
In addition, the presence of large cytoplasmic cellular fragments may also have an impact in the spatial arrangement of the blastomere in the context of ICM and trophectoderm of the blastocyst. Indeed, they can cause apoptosis or the loss of a significant volume of cytoplasm, limiting the rate of blastomere cleavage because of the distortion of the blastomere division planes, leading to abnormal compaction, cavitation and blastocyst formation.
cosmetic embryo microsurgery in terms of removal of fragments and coarse granulation from the embryo before embryo transfer has been suggested to improve cell division and implantation potential
cellular fragments could be indicative of chromosomal problems, and therefore their removal might not improve the potential of many severely fragmented embryos.