Nature sub-journal: reported that scaffolding protein phase separation regulates microbial cell polarity

Cellular asymmetry (also known as cellular polarity) is widely present in animal, plant and microbial cells, and its basic feature is that the mother cell polarizes before division, thereby asymmetrically dividing to produce two daughter cells
with different fates.
Cell polarity is the fundamental cause of diversity in the living world and plays an important role
in cell growth, proliferation, differentiation, development and exercise of cellular functions.
Disturbance of cell polarity is an important manifestation of the development of certain tumors, such as skin cancer; In microorganisms, cellular polarity produces cellular heterogeneity, resulting in resistance tolerance of pathogenic bacteria (e.
g.
, Mycobacterium tuberculosis), environmental resistance, or host immune escape
.

However, due to the high complexity of the regulatory network determined by cell fate, the understanding of cell polarity is in its infancy
.
How cell fate determines how proteins achieve asymmetric subcellular localization in spatiotemporal order is the core problem
to be deeply solved in this field.
As a refined model of asymmetric division of cells, the microbial C.
crescent has unique advantages
in the study of cell polarity.

Recently, the team of Zhao Guoping/Zhao Wei of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, published research results
entitled Phase separation modulates the assembly and dynamics of a polarity-related scaffold-signaling hub in Nature Communication 。 The study found that scaffolding proteins formed polar membrane-free compartments with cell fate determination function through phase separation, revealing a new mechanism
of polar construction and dynamic regulation of microbial cells.

Scaffolding proteins are widely present in cells and typically self-assemble to form macromolecular complexes and recruit customer proteins to specific cell regions, playing a role
in biological processes such as signal transduction, cell division, morphogenesis, and asymmetric division.
Studies have found that in fruit flies and cryptopole nematodes, Par proteins can form membrane-free compartments through phase separation, so that downstream signaling proteins (such as αPKC or PKC3) can be asymmetrically located and distributed, thereby ensuring normal embryonic development
.
This led the team to think
about the possibility of microbial scaffolding proteins regulating cell polarity by phase separation.

The research team used the microorganism of cell development model, Bacillus crescent, as the research object
.
During each cell cycle, Bacillus crescent can divide asymmetrically to produce one flagellar swimmer cell and one stalked daughter cell (Figure 1).

Prior to cell division, the polar localization of a pair of cell fate-determining proteins (phosphorylase PleC and protein kinase DivJ) synergistically regulates phosphorylation levels of multiple downstream signaling proteins and determines the cell fate
of S.
crescent.
It has been shown that the protein kinase DivJ is recruited to the old cell poles
through the scaffolding protein complex of PopZ and SpmX.
Previous studies found that phosphorylase PleC was localized to new cell poles
by a potential scaffolding protein, PodJ.
However, the mechanism of action of microbial scaffolding proteins that recruit cell fate determinants to regulate cell polarity is unclear
.
To this end, the team focused on the PodJ protein and carried out research
on the establishment and regulation mechanism of cell polarity in S.
crescent.

The researchers confirmed the polar localization pattern of PodJ through live-cell time-lapse photography - PodJ specifically recognizes and gathers in the cell neopoles
of Clostella crescent.
At the same time, PodJ was able to be expressed in "distant relative" Escherichia coli and localized to cell polarity
.
The analysis of protein polymer characteristics found that the protein can spontaneously form macromolecular polymers
in vitro.
At the same time, polar aggregation in PodJ in both C.
crescent and Escherichia coli did not depend on other scaffolding proteins, indicating that this protein is a self-assembling scaffolding protein
.

Through PodJ protein sequence analysis, the study predicted that PodJ has a typical intrinsic disordered protein sequence (IDR) and tandem repeat, suggesting the potential for
phase separation of this protein.
Further experiments found that PodJ at endogenous physiological protein levels can spontaneously form micron-sized spherical droplets
in vitro.
At the same time, PodJ can also form protein agglomerates with typical fluidity droplet properties in cells, indicating that phase separation
can occur in vitro and in vivo.

Using the E.
coli heterologous co-expression platform, the research team screened two new PodJ-recruited cell signaling proteins, namely ciliary protein CpaE and flagellin FliG
, in 23 C.
crescent cell cycles or polarity-related proteins.
Confirmatory experiments with S.
crescent showed that the specific recruitment of PodJ to cell signaling functional proteins such as PleC, CpaE and FliG occurred at the cell new pole
.
This suggests that the scaffolding protein PodJ may achieve cell compartmentalization through phase separation and recruit signaling functional proteins
associated with new cell poles.

Further, the research team conducted in-depth mining and characterization
of the structural and functional domains of the PodJ protein.
It was found that both the IDR and CC4-6 domains of PodJ protein could mediate the occurrence
of in vitro phase separation.
To better dissect the domain of PodJ phase separation in vivo, the research team expressed multiple PodJ protein variants in E.
coli, observed their protein localization and accumulation in cells, and analyzed their fluidity
using FRAP.
The results showed that only CC4-6 was able to form cell compartmentalization and exhibit some fluidity
.
This suggests that both CC4-6 and IDR can drive PodJ phase separation in vitro, whereas in cells, PodJ phase separation may rely on CC4-6 and IDR working
together.
The study found that IDR is responsible for recruitment of PleC and CpaE, while CC4-6 is responsible for interaction with FliG by co-expressing signaling functional proteins and PodJ variant proteins in E.
coli
.
This is further confirmed by in vitro phase separation assays for PodJ
.
This suggests that PodJ-driven compartmentalization may directly mediate the recruitment of cell signaling functional proteins, which play a role
in the organization and construction of cell neopolares.

In eukaryotic cells, there is often mutual inhibitory regulation between asymmetric distribution complexes, which is equally important
for homeostatic establishment of cellular polarity.
Studies speculate that similar mechanisms
may exist for the regulation of cellular polarity in prokaryotic cells.
To this end, the research team screened a cell old polar scaffolding protein SpmX among 11 C.
crescent polar proteins and found that it regulated the polar accumulation
of PodJ in E.
coli.
In C.
crescent, the presence of SpmX negatively regulates compartmenttricularization of PodJ cells
.
Further in vitro analysis found that SpmX can directly inhibit the formation of PodJ droplets and can cover the surface of PodJ droplets, thereby inhibiting the growth of droplets and accelerating the aging of droplets, causing damage
to the kinetics and stability of PodJ condensates.
The above in vitro and in vivo observations suggest that the polar remodeling of old and new cells of S.
crescent may be driven
by the inhibitory regulation of PodJ phase separation by SpmX.

This work proposes a new mechanism
for polarity construction and dynamic regulation of microbial cells.
This mechanism involves the assembly of new cell poles and the remodeling of old and new cell poles (Figure 5).

This study elucidates the molecular basis of natural proteins to form membrane-free compartments through phase separation, analyzes the physiological functions and dynamic regulation of cell compartmentalization, and confirms the key role
of phase separation as the assembly of polar hubs of microbial cells and the regulation of asymmetric cell division.
By comparing prokaryotic and eukaryotic cells, the study predicts that phase separation may play a regulatory role
in the development of cell polarity as a general biophysical mechanism.
At the same time, the functional analysis of phase separation and its interaction components is helpful for further artificial design and synthesis of related components, and lays a foundation
for the realization of de novo synthesis of artificial membraneless organelles and single-cell life.

The research work is supported
by the National Natural Science Foundation of China, the National Key R&D Program, the Strategic Leading Science and Technology Project of the Chinese Academy of Sciences, the Basic and Applied Basic Research Fund Committee of Guangdong Province and the Shenzhen Institute of Synthetic Biology Innovation.

Figure 1.
Cell cycle of Bacillus crescent