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.
12/13
Lifeact: a versatile marker to visualize F-actin (Nature method 2010 MPI)
Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues
Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.
Lifeact: a versatile marker to visualize F-actin (Nature method 2010 MPI)
Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues
Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.
12/13
Programming Multicellular Assembly with Synthetic Cell Adhesion Molecules (Nature 2022 UCSF)
a wide array of synthetic cell adhesion molecules (synCAMs) can be generated by combining orthogonal extracellular interactions with intracellular domains from native adhesion molecules, such as cadherins and integrins.
They can be used to mimic the cell-cell interactions with adhesion properties similar to native interactions and then enables rationally programmed assembly of novel multicellular architectures, as well as systematic remodeling of native tissues
Programming Multicellular Assembly with Synthetic Cell Adhesion Molecules (Nature 2022 UCSF)
a wide array of synthetic cell adhesion molecules (synCAMs) can be generated by combining orthogonal extracellular interactions with intracellular domains from native adhesion molecules, such as cadherins and integrins.
They can be used to mimic the cell-cell interactions with adhesion properties similar to native interactions and then enables rationally programmed assembly of novel multicellular architectures, as well as systematic remodeling of native tissues
12/14
Preparation of marked microtubules for the assay of the polarity of microtubule-based motors by fluorescence (Hyman 1991 UCSF)
Short, brightly labeled microtubules can be prepared in two ways
GMPCPP - stable seed
Standard - polymerizing 8 mg/ml rhodamine-labeled tubulin in BRB80 (80 m M K-Pipes, 1 h i m GTP, 1 m M MgCl2, pH 6.8)+ 4 m M MgCl2 +40% glycerol, for 5min
The tubulin aliquot is diluted into BRB80+1 mM GTP to a final concentration of 15 um, and transfered to 37 °C for 1 min
After 1 min, seeds are diluted into the tubulin aliquot at a 1:5 ratio.
The microtubules are allowed to polymerize until they reach a desired length, generally about 15 min.
The microtubules are then stabilized by adding three volumes of BRB80+10 ¿avr taxol and pre-warmed to 37 °C, taking care to avoid shear
The major disadvantage of fluorescence is that it bleaches rapidly, generally within 10 s
Preparation of marked microtubules for the assay of the polarity of microtubule-based motors by fluorescence (Hyman 1991 UCSF)
Short, brightly labeled microtubules can be prepared in two ways
GMPCPP - stable seed
Standard - polymerizing 8 mg/ml rhodamine-labeled tubulin in BRB80 (80 m M K-Pipes, 1 h i m GTP, 1 m M MgCl2, pH 6.8)+ 4 m M MgCl2 +40% glycerol, for 5min
The tubulin aliquot is diluted into BRB80+1 mM GTP to a final concentration of 15 um, and transfered to 37 °C for 1 min
After 1 min, seeds are diluted into the tubulin aliquot at a 1:5 ratio.
The microtubules are allowed to polymerize until they reach a desired length, generally about 15 min.
The microtubules are then stabilized by adding three volumes of BRB80+10 ¿avr taxol and pre-warmed to 37 °C, taking care to avoid shear
The major disadvantage of fluorescence is that it bleaches rapidly, generally within 10 s
12/14
study of Apoptosis In Vitro Using the Xenopus Egg Extract Reconstitution System (Paula Deming and Sally Kornbluth)
study of Apoptosis In Vitro Using the Xenopus Egg Extract Reconstitution System (Paula Deming and Sally Kornbluth)
12/26
DNA content contributes to nuclear size control in Xenopus laevis (2020 MBoC Japan)
These results clearly demonstrate that nuclear expansion is determined not only by cytoplasmic membrane supply but also by the physical properties of chromatin, including DNA quantity and chromatin structure within the nucleus, rather than the coding sequences themselves.
DNA content contributes to nuclear size control in Xenopus laevis (2020 MBoC Japan)
These results clearly demonstrate that nuclear expansion is determined not only by cytoplasmic membrane supply but also by the physical properties of chromatin, including DNA quantity and chromatin structure within the nucleus, rather than the coding sequences themselves.
12/28
Glassfrogs conceal blood in their liver to maintain transparency (Science 2022 Duke)
glass frogs are able to maintain a high level of transparency because a large proportion of their red blood cells are “hidden” in the liver
This strategy allows the frogs to attain transparency when they are the most vulnerable
Glassfrogs conceal blood in their liver to maintain transparency (Science 2022 Duke)
glass frogs are able to maintain a high level of transparency because a large proportion of their red blood cells are “hidden” in the liver
This strategy allows the frogs to attain transparency when they are the most vulnerable
1/14
ATP as a biological hydrotrope (Science 2017 MPI)
Hydrotropes are small molecules that solubilize hydrophobic molecules in aqueous solutions.
ATP at concentrations found in cells could act as a hydrotrope to help solubilize hydrophobic proteins.
ATP inhibits the phase separation of unstructured proteins.
ATP as a biological hydrotrope (Science 2017 MPI)
Hydrotropes are small molecules that solubilize hydrophobic molecules in aqueous solutions.
ATP at concentrations found in cells could act as a hydrotrope to help solubilize hydrophobic proteins.
ATP inhibits the phase separation of unstructured proteins.
1/14
Action at a distance during cytokinesis (JCB 2009 UW & UWM)
cells depleted of astral microtubules conduct accurate, complete cytokinesis
asters alone can support furrow induction without a spindle, but only when sufficiently separated
Ablation of a single centrosome displaces furrows away from the remaining centrosome; ablation of both centrosomes causes broad, inefficient furrowing
the asters confer accuracy and precision to a primary furrow-inducing signal that can reach the cell surface from the spindle without transport on microtubules
astral microtubules are not needed to transmit the cytokinetic signal, even in large round cells where the nearest spindle microtubule is far from the cell surface
Action at a distance during cytokinesis (JCB 2009 UW & UWM)
cells depleted of astral microtubules conduct accurate, complete cytokinesis
asters alone can support furrow induction without a spindle, but only when sufficiently separated
Ablation of a single centrosome displaces furrows away from the remaining centrosome; ablation of both centrosomes causes broad, inefficient furrowing
the asters confer accuracy and precision to a primary furrow-inducing signal that can reach the cell surface from the spindle without transport on microtubules
astral microtubules are not needed to transmit the cytokinetic signal, even in large round cells where the nearest spindle microtubule is far from the cell surface
1/14
A mitotic chromatin phase transition prevents perforation by microtubules (Nature 2022 Australia)
a substantial global reduction in chromatin solubility caused by deacetylation during mitotic entry converts chromosomes into phase-separated bodies rather than loose bottlebrush structures with chromatin fibre loops extending into the cytoplasm
The immiscible mitotic chromatin forms a surface that provides resistance to microtubule perforation while allowing local chromatin fibre sliding internally, as required for continuous dynamic loop formation by condensin
By contrast, hyperacetylated mitotic chromosomes lack a defined surface boundary, are frequently perforated by microtubules and are prone to missegregation.
A mitotic chromatin phase transition prevents perforation by microtubules (Nature 2022 Australia)
a substantial global reduction in chromatin solubility caused by deacetylation during mitotic entry converts chromosomes into phase-separated bodies rather than loose bottlebrush structures with chromatin fibre loops extending into the cytoplasm
The immiscible mitotic chromatin forms a surface that provides resistance to microtubule perforation while allowing local chromatin fibre sliding internally, as required for continuous dynamic loop formation by condensin
By contrast, hyperacetylated mitotic chromosomes lack a defined surface boundary, are frequently perforated by microtubules and are prone to missegregation.
1/14
Prc1E and Kif4A control microtubule organization within and between large Xenopus egg asters (2018 MBoC Tim Mitchison)
conserved cytokinesis midzone proteins: Prc1 and Kif4A. Prc1E
Prc1E was required for Kif4A recruitment but not vice versa
plus-end growth slowed and terminated preferentially within interaction zones, resulting in a block to interpenetration that depended on both Prc1E and Kif4A
We propose that Prc1E and Kif4, together with catastrophe factors, promote “anti-parallel pruning” that enforces radial organization within asters and generates boundaries to microtubule growth between asters.
The plus-end–directed kinesin-family motor Kif23 (a.k.a. MKLP1) transports the furrow-stimulating factor RacGap1 (together they constitute the centralspindlin complex) to the center of the midzone, while the plus-end–directed motor Kif20A (a.k.a. MKLP2) transports the chromosome passenger complex (CPC) - Centralspindlin and the CPC then signal locally to the equatorial cortex to initiate the furrow
Prc1E and Kif4A control microtubule organization within and between large Xenopus egg asters (2018 MBoC Tim Mitchison)
conserved cytokinesis midzone proteins: Prc1 and Kif4A. Prc1E
Prc1E was required for Kif4A recruitment but not vice versa
plus-end growth slowed and terminated preferentially within interaction zones, resulting in a block to interpenetration that depended on both Prc1E and Kif4A
We propose that Prc1E and Kif4, together with catastrophe factors, promote “anti-parallel pruning” that enforces radial organization within asters and generates boundaries to microtubule growth between asters.
The plus-end–directed kinesin-family motor Kif23 (a.k.a. MKLP1) transports the furrow-stimulating factor RacGap1 (together they constitute the centralspindlin complex) to the center of the midzone, while the plus-end–directed motor Kif20A (a.k.a. MKLP2) transports the chromosome passenger complex (CPC) - Centralspindlin and the CPC then signal locally to the equatorial cortex to initiate the furrow
1/15
In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers (2018 Jove)
Escherichia coli MinCDE system (a reaction-diffusion system) - This system is based on the ATPase MinD, the ATPase activating protein MinE, and the membrane as a spatial reaction matrix; MinC is not part of the pattern formation mechanism, but is the actual functional agent: an inhibitor of the main divisome protein FtsZ
In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers (2018 Jove)
Escherichia coli MinCDE system (a reaction-diffusion system) - This system is based on the ATPase MinD, the ATPase activating protein MinE, and the membrane as a spatial reaction matrix; MinC is not part of the pattern formation mechanism, but is the actual functional agent: an inhibitor of the main divisome protein FtsZ