Editor
Rajesh Ranjan
  • Research Specialist II, Howard Hughes Medical institute; Department of Biology, Johns Hopkins University, Baltimore, MD
Research fields
  • Cell Biology, Developmental Biology, Molecular Biology, Stem Cell
Quantitative Analysis of Kinetochore Protein Levels and Inter-Kinetochore Distances in Mammalian Cells During Mitosis

The mammalian kinetochore is a multi-layered protein complex that forms on the centromeric chromatin. The kinetochore serves as the attachment hub for the plus ends of microtubules emanating from the centrosomes during mitosis. For karyokinesis, bipolar kinetochore-microtubule attachment and subsequent microtubule depolymerization lead to the development of inter-kinetochore tension between the sister chromatids. These events are instrumental in initiating a signaling cascade culminating in the segregation of the sister chromatids equally between the new daughter cells. Of the hundreds of conserved proteins that constitute the mammalian kinetochore, many that reside in the outermost layer are loaded during early mitosis and removed around metaphase-anaphase. Dynamically localized kinetochore proteins include those required for kinetochore-microtubule attachment, spindle assembly checkpoint proteins, various kinases, and molecular motors. The abundance of these kinetochore-localized proteins varies at prometaphase, metaphase, and anaphase, and is thus considered diagnostic of the fidelity of progression through these stages of mitosis. Here, we document detailed, state-of-the-art methodologies based on high-resolution fluorescence confocal microscopy followed by quantification of the levels of kinetochore-localized proteins during mitosis. We also document methods to accurately measure distances between sister kinetochores in mammalian cells, a surrogate readout for inter-kinetochore tension, which is essential for chromosome segregation.

Efficient Gene-Editing in Human Pluripotent Stem Cells Through Simplified Assembly of Adeno-Associated Viral (AAV) Donor Templates

Gene-edited human pluripotent stem cells provide attractive model systems to functionally interrogate the role of specific genetic variants in relevant cell types. However, the need to isolate and screen edited clones often remains a bottleneck, in particular when recombination rates are sub-optimal. Here, we present a protocol for flexible gene editing combining Cas9 ribonucleoprotein with donor templates delivered by adeno-associated virus (AAV) vectors to yield high rates of homologous recombination. To streamline the workflow, we designed a modular system for one-step assembly of targeting vectors based on Golden Gate cloning and developed a rapid protocol for small-scale isolation of AAV virions of serotype DJ. High homology-directed repair (HDR) rates in human pluripotent stem cells (hPSCs), ~70% in ACTB and ~30% in LMNB1, were achieved using this approach, also with short (300 bp) homology arms. The modular design of donor templates is flexible and allows for the generation of conditional and/or complex alleles. This protocol thus provides a flexible and efficient strategy workflow to rapidly generate gene-edited hPSC lines.

Dissecting the Mechanical Control of Mitotic Entry Using a Cell Confinement Setup
Authors:  Margarida Dantas, Débora Vareiro and Jorge G. Ferreira, date: 03/20/2024, view: 1489, Q&A: 0

Proliferating cells need to cope with extensive cytoskeletal and nuclear remodeling as they prepare to divide. These events are tightly regulated by the nuclear translocation of the cyclin B1-CDK1 complex, that is partly dependent on nuclear tension. Standard experimental approaches do not allow the manipulation of forces acting on cells in a time-resolved manner. Here, we describe a protocol that enables dynamic mechanical manipulation of single cells with high spatial and temporal resolution and its application in the context of cell division. In addition, we also outline a method for the manipulation of substrate stiffness using polyacrylamide hydrogels. Finally, we describe a static cell confinement setup, which can be used to study the impact of prolonged mechanical stimulation in populations of cells.


Key features

• Protocol for microfabrication of confinement devices.

• Single-cell dynamic confinement coupled with high-resolution microscopy.

• Static cell confinement protocol that can be combined with super-resolution STED microscopy.

• Analysis of the mechanical control of mitotic entry in a time-resolved manner.


Graphical overview


Methodology to Create Auxin-Inducible Degron Tagging System to Control Expression of a Target Protein in Mammalian Cell Lines
Authors:  Amit Rahi, Deepika K. Sodhi, Christine B. Magdongon, Rajina Shakya and Dileep Varma, date: 01/20/2024, view: 1327, Q&A: 0

The auxin-inducible degron (AID) system is a versatile tool in cell biology and genetics, enabling conditional protein regulation through auxin-induced degradation. Integrating CRISPR/Cas9 with AID expedites tagging and depletion of a required protein in human and mouse cells. The mechanism of AID involves interactions between receptors like TIR1 and the AID tag fused to the target protein. The presence of auxin triggers protein ubiquitination, leading to proteasome-mediated degradation. We have used AID to explore the mitotic functions of the replication licensing protein CDT1. Swift CDT1 degradation via AID upon auxin addition achieves precise mitotic inhibition, revealing defects in mitotic spindle structure and chromosome misalignment. Using live imaging, we found that mitosis-specific degradation of CDT1 delayed progression and chromosome mis-segregation. AID-mediated CDT1 inhibition surpasses siRNA-based methods, offering a robust approach to probe CDT1’s mitotic roles. The advantages of AID include targeted degradation and temporal control, facilitating rapid induction and reversal of degradation—contrasting siRNA’s delayed RNA degradation and protein turnover. In summary, the AID technique enhances precision, control, and efficiency in studying protein function and regulation across diverse cellular contexts. In this article, we provide a step-by-step methodology for generating an efficient AID-tagging system, keeping in mind the important considerations that need to be adopted to use it for investigating or characterizing protein function in a temporally controlled manner.


Key features

• The auxin-inducible degron (AID) system serves as a versatile tool, enabling conditional protein regulation through auxin-induced degradation in cell biology and genetics.

• Integration of CRISPR/Cas9 knock-in technology with AID expedites the tagging and depletion of essential proteins in mammalian cells.

• AID’s application extends to exploring the mitotic functions of the replication licensing protein CDT1, achieving precise mitotic inhibition and revealing spindle defects and chromosome misalignment.

• The AID system and its diverse applications advance the understanding of protein function and cellular processes, contributing to the study of protein regulation and function.


Graphical overview



Cdt1–auxin-inducible degron (AID) tagging workflow. (A) Schematic of the cloned Cdt1 gRNA vector and the repair template generated to endogenously tag the Cdt1 genomic locus with YFP and AID at the C-terminal using CRISPR/CAS9-based genome editing. The two plasmids are transfected into DLD1-TIR1 stable cells, followed by sorting and scaling up of YFP-positive single cells. (B) The molecular mechanism of auxin-induced proteasome-mediated degradation of the target protein (CDT1) shown at the bottom of the figure is well worked out.

Simultaneous Profiling of Chromosome Conformation and Gene Expression in Single Cells
Authors:  Yujie Chen, Heming Xu, Zhiyuan Liu and Dong Xing, date: 11/20/2023, view: 980, Q&A: 0

Rapid development in single-cell chromosome conformation capture technologies has provided valuable insights into the importance of spatial genome architecture for gene regulation. However, a long-standing technical gap remains in the simultaneous characterization of three-dimensional genomes and transcriptomes in the same cell. We have described an assay named Hi-C and RNA-seq employed simultaneously (HiRES), which integrates in situ reverse transcription and chromosome conformation capture (3C) for the parallel analysis of chromatin organization and gene expression. Here, we provide a detailed implementation of the assay, using mouse embryos and cerebral cortices as examples. The versatility of this method extends beyond these two samples, with the potential to be used in various other cell types.


Key features

• A multi-omics sequencing approach to profile 3D genome structure and gene expression simultaneously in single cells.

• Compatible with animal tissues.

• One-tube amplification of both DNA and RNA components.

• Requires three days to complete.


Graphical overview



Schematic illustration for the Hi-C and RNA-seq employed simultaneously (HiRES) workflow
Differentiation of Bone Marrow Monocytes into Alveolar Macrophages-like Cells through Co-culture with Lung Epithelial Cells and Group 2 Innate Lymphoid Cells
Authors:  Pauline Loos, Thomas Marichal, Bénédicte Machiels and Laurent Gillet, date: 09/20/2023, view: 826, Q&A: 0

During life, the embryonic alveolar macrophage (AM) population undergoes successive waves of depletion and replenishment in response to infectious and inflammatory episodes. While resident AMs are traditionally described as from embryonic origin, their ontogeny following inflammation or infection is much more complex. Indeed, it appears that the contribution of monocytes (MOs) to the AM pool is variable and depends on the type of inflammation, its severity, and the signals released in the microenvironment of the pulmonary niche (peripheral imprinting) and/or in the bone marrow (central imprinting). Deciphering the cellular and molecular mechanisms regulating the differentiation of MOs into AMs remains an area of intense investigation, as this could potentially explain part of the inter-individual susceptibility to respiratory immunopathologies. Here, we detail a relevant ex vivo co-culture model to investigate how lung epithelial cells (ECs) and group 2 lung innate lymphoid cells (ILC2s) contribute to the differentiation of recruited MOs into AMs. Interestingly, the presence of lung ILC2s and ECs provides the necessary niche signals to ensure the differentiation of bone marrow MOs into AMs, thus establishing an accessible model to study the underlying mechanisms following different infection or inflammation processes.


Key features

• Ex vivo co-culture model of the alveolar niche.

• Deciphering the particular niche signals underlying the differentiation of MO into AMs and their functional polarization.


Graphical overview
This protocol described the isolation of bone marrow monocytes (MOs), lung epithelial cells (ECs), and lung group 2 lung innate lymphoid cells (ILC2s) and the ex vivo co-culture of these cells to drive the differentiation of bone marrow MOs into alveolar macrophages (AMs).




This co-culture experiment is composed of three steps (Graphical overview):
1. Identification and FACS-sorting of ECs and MOs isolated from the lung and the bone marrow of naive mice, respectively.
2. Culture of these ECs and bone marrow MOs for three days.
3. Addition of ILC2s isolated from the lung of naïve mice or mice subjected to a treatment/infection of interest.

Optogenetic Induction of Pyroptosis, Necroptosis, and Apoptosis in Mammalian Cell Lines
Authors:  Kateryna Shkarina and Petr Broz, date: 07/20/2023, view: 1232, Q&A: 0

Regulated cell death plays a key role in immunity, development, and homeostasis, but is also associated with a number of pathologies such as autoinflammatory and neurodegenerative diseases and cancer. However, despite the extensive mechanistic research of different cell death modalities, the direct comparison of different forms of cell death and their consequences on the cellular and tissue level remain poorly characterized. Comparative studies are hindered by the mechanistic and kinetic differences between cell death modalities, as well as the inability to selectively induce different cell death programs in an individual cell within cell populations or tissues. In this method, we present a protocol for rapid and specific optogenetic activation of three major types of programmed cell death: apoptosis, necroptosis, and pyroptosis, using light-induced forced oligomerization of their major effector proteins (caspases or kinases).

Preparation of Human Kidney Progenitor Cultures and Their Differentiation into Podocytes

Kidney diseases are a global health concern. Modeling of kidney disease for translational research is often challenging because of species specificities or the postmitotic status of kidney epithelial cells that make primary cultures, for example podocytes. Here, we report a protocol for preparing primary cultures of podocytes based on the isolation and in vitro propagation of immature kidney progenitor cells subsequently differentiated into mature podocytes. This protocol can be useful for studying physiology and pathophysiology of human kidney progenitors and to obtain differentiated podocytes for modeling podocytopathies and other kidney disorders involving podocytes.


Graphical overview


Ex vivo Drug Sensitivity Imaging-based Platform for Primary Acute Lymphoblastic Leukemia Cells

Resistance of acute lymphoblastic leukemia (ALL) cells to chemotherapy, whether present at diagnosis or acquired during treatment, is a major cause of treatment failure. Primary ALL cells are accessible for drug sensitivity testing at the time of new diagnosis or at relapse, but there are major limitations with current methods for determining drug sensitivity ex vivo. Here, we describe a functional precision medicine method using a fluorescence imaging platform to test drug sensitivity profiles of primary ALL cells. Leukemia cells are co-cultured with mesenchymal stromal cells and tested with a panel of 40 anti-leukemia drugs to determine individual patterns of drug resistance and sensitivity (“pharmacotype”). This imaging-based pharmacotyping assay addresses the limitations of prior ex vivo drug sensitivity methods by automating data analysis to produce high-throughput data while requiring fewer cells and significantly decreasing the labor-intensive time required to conduct the assay. The integration of drug sensitivity data with genomic profiling provides a basis for rational genomics-guided precision medicine.


Key features

• Analysis of primary acute lymphoblastic leukemia (ALL) blasts obtained at diagnosis from bone marrow aspirate or peripheral blood.

• Experiments are performed ex vivo with mesenchymal stromal cell co-culture and require four days to complete.

• This fluorescence imaging–based protocol enhances previous ex vivo drug sensitivity assays and improves efficiency by requiring fewer primary cells while increasing the number of drugs tested to 40.

• It takes approximately 2–3 h for sample preparation and processing and a 1.5-hour imaging time.


Graphical overview




BM: bone marrow; PB: peripheral blood; ALL: acute lymphoblastic leukemia; MNCs: mononuclear cells, which include leukemia cells when present; MSCs: mesenchymal stromal cells; LC50: drug concentration that kills 50% of the leukemia cells

A New Approach to Generate Gastruloids to Develop Anterior Neural Tissues
Authors:  Mehmet Girgin, Nicolas Broguiere, Lorenzo Mattolini and Matthias Lutolf, date: 07/20/2023, view: 1202, Q&A: 0

Embryonic development is a complex process integrating cell fate decisions and morphogenesis in a spatiotemporally controlled manner. Previous studies with model organisms laid the foundation of our knowledge on post-implantation development; however, studying mammalian embryos at this stage is a difficult and laborious process. Early attempts to recapitulate mammalian development in vitro begun with embryoid bodies (EBs), in which aggregates of mouse embryonic stem cells (mESCs) were shown to differentiate into spatially arranged germ layers. A more revised version of EBs, gastruloids, improved the germ layer differentiation efficiency and demonstrated cell fate patterning on multiple axes. However, gastruloids lack anterior neural progenitors that give rise to brain tissues in the embryo. Here, we report a novel culture protocol to coax mESCs into post-implantation epiblast-like (EPI) aggregates in high throughput on bioengineered microwell arrays. We show that upon inhibition of the Wnt signaling pathway, EPI aggregates establish an extended axial patterning, leading to co-derivation of anterior neural progenitors and posterior tissues. Our approach is amenable to large-scale studies aimed at identifying novel regulators of gastrulation and anterior neural development that is currently out of reach with existing embryoid models. This work should contribute to the advancement of the nascent field of synthetic embryology, opening up exciting perspectives for various applications of pluripotent stem cells in disease modeling and tissue engineering.


Key features

• A new gastruloid culture system to model post-implantation mouse embryonic development in vitro

• High-throughput formation of epiblast-like aggregates on hydrogel microwells

• Builds upon conventional gastruloid cultures and provides insight into the role of Wnt signaling for the formation of anterior neural tissues


Graphical overview


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